Recombinant microorganism for the production of useful metabolites

ABSTRACT

Described are recombinant microorganisms characterized by having phosphoketolase activity, having a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s) encoding phosphofructokinase or by reducing phosphofructokinase activity as compared to a non-modified microorganism and having a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism. These microorganisms can be used for the production of useful metabolites such as acetone, isobutene or propene.

This Application is a continuation of Ser. No. 14/232,011, filed on May21, 2014, which is a 371 National Phase Application of PCT/EP2012/063685filed Jul. 12, 2012 and which claims priority to EP 111735635 filed onJul. 12, 2011. The entire contents of Ser. No. 14/232,011,PCT/EP2012/063685 and EP 111735635 are hereby incorporated by referencein their entirety.

The present invention relates to a recombinant microorganismcharacterized by having phosphoketolase activity, having a diminished orinactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of thegene(s) encoding the phosphofructokinase or by reducing thephosphofructokinase activity as compared to a non-modified microorganismor not possessing phosphofructokinase activity and having a diminishedor inactivated oxidative branch of the pentose phosphate pathway (PPP)by inactivation of the gene(s) encoding the glucose-6-phosphatedehydrogenase or by reducing the glucose-6-phosphate dehydrogenaseactivity as compared to a non-modified microorganism or not possessingglucose-6-phosphate dehydrogenase activity. Such a microorganism can beused for the production of useful metabolites such as acetone, isobuteneor propene.

For the past several decades, practitioners of metabolic engineeringhave endeavoured to provide biological solutions for the production ofchemicals, thus, providing alternatives to more traditional chemicalprocesses. In general, biological solutions allow for the utilization ofrenewable feedstocks (e.g. sugars) and compete with existingpetrochemical based processes. A multi-step, biological solution for theproduction of a chemical typically comprises a microorganism as thecatalyst for the conversion of feedstock to a target molecule. Acomplete set of enzyme reactions for the production of a particulartarget molecule can be grouped into those belonging to central carbonpathways and those belonging to the product specific pathway. Thereactions belonging to central carbon and product specific pathways arelinked in that redox (typically, NAD(P)H) and energetic (typically, ATP)constraints of each and every enzyme reaction must be accounted for inan overall balance contributing to the competitiveness of the process.Historically, central carbon pathways of heterotrophs growing on sugarshave been described as the Embden-Meyerhoff-Parnas pathway (EMPP), thepentose phosphate pathway (PPP), the Entner-Doudoroff pathway (EDP), andthe phosphoketolase pathway (PKP) (see Gottschalk (1986), BacterialMetabolism, 2^(nd) Edition, Springer-Verlag, New York). Each centralpathway or combinations of central pathways offer advantages anddisadvantages with respect to a specific target molecule. In order toprovide competitive bioprocesses, recombinant microorganisms withmodifications involving the EMPP, PPP and EDP have been described (M.Emmerling et al., Metab. Eng. 1:117 (1999); L. O. Ingram et al., Appl.Environ. Microbiol. 53: 2420 (1987); C. T. Trinh et al., Appl. Environ.Microbiol. 74:3634 (2008)). More recently, recombinant microorganismswith modifications involving the PKP have been described (seeSonderegger et al. Appl. Environ. Microbiol. 70 (2004), 2892-2897, U.S.Pat. No. 7,253,001, Chinen et al. J. Biosci. Bioeng. 103 (2007),262-269, U.S. Pat. No. 7,785,858; Fleige et al., Appl. Microbiol. CellPhysiol. 91 (2011), 769-776).

The EMPP converts 1 mol glucose to 2 mol pyruvate (PYR). When acetyl-CoAis desired, 1 mol PYR can be converted to 1 mol of acetyl-CoA with theconcomitant generation of 1 mol CO₂ and 1 mol NADH. The sum of thereactions is given in Equation 1.glucose+2ADP+2H₃PO₄+2CoA+4NAD⁺→2acetyl-CoA+2CO₂+2ATP+2H₂O+4NADH+4H⁺  (Equation 1)

The PPP provides a means to convert 1 mol glucose to 1 mol CO₂ and 2 molNADPH, with the concomitant generation of 0.67 mol fructose-6-phosphat(F6P) and 0.33 mol glyceraldehyde-3-phosphate (GAP). The F6P and GAPthus formed must be metabolized by other reaction pathways, e.g. by theEMPP. The EDP converts 1 mol glucose to 1 mol GAP and 1 mol PYR with theconcomitant generation of 1 mol NADPH. As with the PPP, the GAP thusformed must be metabolized by other reaction pathways. The PKP providesa means to convert 1 mol glucose to 1 mol GAP and 1.5 mol acetylphosphate (AcP). When acetyl-CoA is desired, 1 equivalent of AcP plus 1equivalent coenzyme A (CoA) can be converted to 1 equivalent acetyl-CoAand 1 equivalent inorganic phosphate (Pi) by the action ofphosphotransacetylase.

For specific target molecules derived from AcCoA moieties generatedthrough the PKP and near redox neutrality to the AcCoA moieties, thereexists a deficiency in the overall energy balance. The PKP (and,similarly, the PPP and EDP) does not generate ATP for the conversion ofglucose to glucose-6-phosphate. In the case of phosphoenolpyruvate(PEP)-dependent glucose uptake, PEP must be generated by other means,e.g. through the EMPP. Recycling GAP through the PKP exacerbates theissue, particularly when the product specific pathway provides littleATP.

Sonderegger (loc. cit.) and U.S. Pat. No. 7,253,001 disclose recombinantSaccharomyces cerevisiae strains comprising native or overexpressedphosphoketolase activity together with overexpressedphosphotransacetylase to increase the yield in the conversion ofglucose/xylose mixtures to ethanol. These strains featurePEP-independent glucose uptake with both the EMPP and the PPP operative.

Chinen (loc. cit.) and U.S. Pat. No. 7,785,858 disclose a recombinantbacterium selected from the group consisting of the Enterobacteriaceaefamiliy, Coryneform bacterium, and Bacillus bacterium comprisingincreased phosphoketolase activity for the conversion of glucose totarget molecules which are produced via the intermediate acetyl-CoA,including the group consisting of L-glutamic acid, L-glutamine,L-proline, L-arginine, L-leucine, L-cysteine, succinate andpolyhydroxybutyrate. These strains feature PEP-dependent glucose uptakewith the EMPP operative. Notably, the activity of phosphofructokinase inthe bacterium of U.S. Pat. No. 7,785,858 is reduced compared to that ofa wild-type or non-modified strain (see page 33).

Whether a particular microorganism utilizes PEP-independent glucoseuptake or PEP-dependent glucose uptake impacts the overall energeticbalance of a process. For example, S. cerevisiae strains naturallyemploy PEP-independent glucose uptake while Escherichia coli strainsnaturally employ PEP-dependent glucose uptake. E. coli strains have beendisclosed where PEP-dependent glucose uptake has been replaced withPEP-independent glucose uptake. Flores et al. (Metabolic Engineering(2005) 7, 70-87) and U.S. Pat. No. 7,371,558. In particular, U.S. Pat.No. 7,371,558 discloses the glucose uptake modification to increase theyield in the conversion of glucose to 1,3-propanediol. The strainsfeature PEP-independent glucose uptake with both the EMPP and the PPPoperative, notably with no phosphoketolase activity present.

There is a need to develop recombinant microorganisms, comprisingcentral carbon and product specific pathways that maximize theconversion of feedstock to product by best accommodating the redox andenergetic constraints of enzyme reactions. Applicants have addressedthis need by providing the embodiments as defined in the claims.

Thus, the present invention relates to a recombinant microorganismcharacterized by:

-   a) having phosphoketolase activity;-   b) (i) having a diminished or inactivated Embden-Meyerhof-Parnas    pathway (EMPP) by inactivation of the gene(s) encoding    phosphofructokinase or by reducing phosphofructokinase activity as    compared to a non-modified microorganism; or    -   (ii) not possessing phosphofructokinase activity        and-   c) (i) having a diminished or inactivated oxidative branch of the    pentose phosphate pathway (PPP) by inactivation of the gene(s)    encoding glucose-6-phosphate dehydrogenase or by reducing    glucose-6-phosphate dehydrogenase activity as compared to a    non-modified microorganism; or    -   (ii) not possessing glucose-6-phosphate dehydrogenase activity.

The microorganism according to the present invention is characterised byhaving phosphoketolase activity, so as to increase the flux ofacetyl-CoA produced. Usually, a microorganism converts glucose via theEmbden-Meyerhof-Parnas pathway into pyruvate which can then be convertedinto acetyl-CoA by the enzyme pyruvate dehydrogenase. However, thisconversion is accompanied by the release of CO₂ and, thus, one carbonatom is lost which might have been used in the production of usefulmetabolites. In order to increase the amount of acetyl-CoA in amicroorganism it is therefore desirable that acetyl-CoA is formed via adifferent pathway to avoid the loss of carbon atoms. By using amicroorganism having phosphoketolase activity, phosphate andfructose-6-phosphate are converted to erythrose-4-phosphate andacetylphosphate and the phosphotransacetylase further convertsacetylphosphate into acetyl-CoA without loss of a carbon atom. Thus, inthe end, the yield of acetyl-CoA can be increased by using amicroorganism having phosphoketolase activity. Such a microorganism iscapable of converting glucose into acetyl-CoA without loss of a carbonatom. Recombinant microorganisms in which a phosphoketolase is naturallyor heterologously expressed are disclosed in U.S. Pat. No. 7,785,858 andU.S. Pat. No. 7,253,001.

The term “phosphoketolase activity” as used in the present inventionmeans an enzymatic activity that is capable of convertingD-xylulose-5-phosphate into D-glyceraldehyde-3-phosphate according tothe following reaction:D-xylulose-5-phosphate+phosphate→D-glyceraldehyde-3-phosphate+acetylphosphate+wateror that is capable to catalyze the above shown reaction and that is alsoable to convert D-fructose-6-phosphate to D-erythrose-4-phosphateaccording to the following reaction:D-Fructose 6-phosphate+phosphate→acetyl phosphate+D-erythrose4-phosphate+water

The former phosphoketolases are usually classified in EC 4.1.2.9 and thelatter in EC 4.1.2.22. Both types of phosphoketolases can be employed inthe scope of the present invention. FIG. 1 shows schemes for the overallreactions using the two options of the phosphoketolase as describedherein.

This enzymatic activity can be measured by assays known in the art. Anexample for such an assay is given in the Example section below.

In the context of the present invention, a microorganism which hasphosphoketolase activity can, e.g., be a microorganism which naturallyhas phosphoketolase activity or a microorganism that does not naturallyhave phosphoketolase activity and has been genetically modified toexpress a phosphoketolase or a microorganism which naturally hasphosphoketolase activity and which has been genetically modified, e.g.transformed with a nucleic acid, e.g. a vector, encoding aphosphoketolase in order to increase the phosphoketolase activity insaid microorganism.

Microorganisms that inherently, i.e. naturally, have phosphoketolaseactivity are known in the art and any of them can be used in the contextof the present invention.

It is also possible in the context of the present invention that themicroorganism is a microorganism which naturally does not havephosphoketolase activity but which is genetically modified so as tocomprise a nucleotide sequence allowing the expression of aphosphoketolase. Similarly, the microorganism may also be amicroorganism which naturally has phosphoketolase activity but which isgenetically modified so as to enhance the phosphoketolase activity, e.g.by the introduction of an exogenous nucleotide sequence encoding aphosphoketolase.

The genetic modification of microorganisms to express an enzyme ofinterest will be described in detail below.

The phosphoketolase expressed in the microorganism according to theinvention can be any phosphoketolase, in particular a phosphoketolasefrom prokaryotic or eukaryotic organisms. Prokaryotic phosphoketolasesare described, e.g., from Lactococcus lactis and an example is given inthe Example section.

In a preferred embodiment of the present invention the phosphoketolaseis an enzyme comprising an amino acid sequence as encoded by SQ0005shown in the Example section or a sequence which is at least n %identical to that amino acid sequence and having the activity of aphosphoketolase with n being an integer between 10 and 100, preferably10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99.

Preferably, the degree of identity is determined by comparing therespective sequence with the amino acid sequence of any one of theabove-mentioned SEQ ID NOs. When the sequences which are compared do nothave the same length, the degree of identity preferably either refers tothe percentage of amino acid residues in the shorter sequence which areidentical to amino acid residues in the longer sequence or to thepercentage of amino acid residues in the longer sequence which areidentical to amino acid residues in the shorter sequence. The degree ofsequence identity can be determined according to methods well known inthe art using preferably suitable computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particularsequence is, for instance, 80% identical to a reference sequence defaultsettings may be used or the settings are preferably as follows: Matrix:blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delaydivergent: 40; Gap separation distance: 8 for comparisons of amino acidsequences. For nucleotide sequence comparisons, the Extend gap penaltyis preferably set to 5.0.

Preferably, the degree of identity is calculated over the completelength of the sequence.

The phosphoketolase expressed in the microorganism according to theinvention can be a naturally occurring phosphoketolase or it can be aphosphoketolase which is derived from a naturally occurringphosphoketolase, e.g. by the introduction of mutations or otheralterations which, e.g., alter or improve the enzymatic activity, thestability, etc.

Methods for modifying and/or improving the desired enzymatic activitiesof proteins are well-known to the person skilled in the art and include,e.g., random mutagenesis or site-directed mutagenesis and subsequentselection of enzymes having the desired properties or approaches of theso-called “directed evolution”.

For example, for genetic modification in prokaryotic cells, a nucleicacid molecule encoding phosphoketolase can be introduced into plasmidswhich permit mutagenesis or sequence modification by recombination ofDNA sequences. Standard methods (see Sambrook and Russell (2001),Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor,N.Y., USA) allow base exchanges to be performed or natural or syntheticsequences to be added. DNA fragments can be ligated by using adaptersand linkers complementary to the fragments. Moreover, engineeringmeasures which provide suitable restriction sites or remove surplus DNAor restriction sites can be used. In those cases, in which insertions,deletions or substitutions are possible, in vitro mutagenesis, “primerrepair”, restriction or ligation can be used. In general, a sequenceanalysis, restriction analysis and other methods of biochemistry andmolecular biology are carried out as analysis methods. The resultingphosphoketolase variants are then tested for the desired activity, e.g.,enzymatic activity, with an assay as described above and in particularfor their increased enzyme activity.

As described above, the microorganism of the invention may be amicroorganism which has been genetically modified by the introduction ofa nucleic acid molecule encoding a phosphoketolase. Thus, in a preferredembodiment, the recombinant microorganism is a recombinant microorganismwhich has been genetically modified to have an increased phosphoketolaseactivity. This can be achieved e.g. by transforming the microorganismwith a nucleic acid encoding a phosphoketolase. A detailed descriptionof genetic modification of microorganisms will be given further below.Preferably, the nucleic acid molecule introduced into the microorganismis a nucleic acid molecule which is heterologous with respect to themicroorganism, i.e. it does not naturally occur in said microorganism.

In the context of the present invention, an “increased activity” meansthat the expression and/or the activity of an enzyme, in particular ofthe phosphoketolase in the genetically modified microorganism is atleast 10%, preferably at least 20%, more preferably at least 30% or 50%,even more preferably at least 70% or 80% and particularly preferred atleast 90% or 100% higher than in the corresponding non-modifiedmicroorganism. In even more preferred embodiments the increase inexpression and/or activity may be at least 150%, at least 200% or atleast 500%. In particularly preferred embodiments the expression is atleast 10-fold, more preferably at least 100-fold and even more preferredat least 1000-fold higher than in the corresponding non-modifiedmicroorganism.

The term “increased” expression/activity also covers the situation inwhich the corresponding non-modified microorganism does not express acorresponding enzyme, e.g. phosphoketolase, so that the correspondingexpression/activity in the non-modified microorganism is zero.

Methods for measuring the level of expression of a given protein in acell are well known to the person skilled in the art. In one embodiment,the measurement of the level of expression is done by measuring theamount of the corresponding protein. Corresponding methods are wellknown to the person skilled in the art and include Western Blot, ELISAetc. In another embodiment the measurement of the level of expression isdone by measuring the amount of the corresponding RNA. Correspondingmethods are well known to the person skilled in the art and include,e.g., Northern Blot.

Methods for measuring the enzymatic activity of the phosphoketolase areknown in the art and have already been described above.

The microorganism according to the present invention is furthercharacterised by having a diminished or inactivatedEmbden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s)encoding a phosphofructokinase or by reducing the phosphofructokinaseactivity as compared to a non-modified microorganism or by notpossessing phosphofructokinase activity. Thus, the microorganism iseither a microorganism which naturally has an EMPP includingphosphofructokinase activity but which has been modified, in particulargenetically modified, so that the phosphofructokinase activity is eithercompletely abolished or so that it is reduced compared to thecorresponding non-modified microorganism, or the microorganism is amicroorganism which naturally does not possess a phosphofructokinaseactivity.

As already mentioned above, when glucose is processed via the EMPP toacetyl-CoA, one carbon atom is lost by the release of CO₂ in the laststep. By introducing the phosphoketolase, this loss can be avoided.Since fructose-6-phosphate is a substrate for the phosphoketolase, it isdesirable that the pool of fructose-6-phosphate is kept at a high levelin the microorganism in order to increase the yield in acetyl-CoA. Sincefructose-6-phosphate is also a substrate for an enzyme of theEmbden-Meyerhof-Parnas pathway, i.e. the phosphofructokinase, therecombinant microorganism of the present invention has a reducedphosphofructokinase activity as compared to a non-modified microorganismor the gene(s) encoding a phosphofructokinase has/have been inactivated.This ensures the flux of fructose-6-phosphate is directed to thephosphoketolase and to the production of acetyl-CoA without loss of CO₂because fructose-6-phosphate or most of fructose-6-phosphate can nolonger be processed via the Embden-Meyerhof-Parnas pathway. Recombinantmicroorganisms in which a phosphoketolase is naturally or heterologouslyexpressed and which have reduced phosphofructokinase activity aredisclosed in U.S. Pat. No. 7,785,858.

The “phosphofructokinase activity” means an enzymatic activity thatconverts ATP and fructose-6-phosphate to ADP andfructose-1,6-bisphosphate (EC 2.7.1.11). This enzymatic activity can bemeasured by assays known in the art as, for example, described byKotlarz et al. (Methods Enzymol. (1982) 90, 60-70).

The term “a microorganism which is characterised by having a diminishedor inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation ofthe gene(s) encoding a phosphofructokinase or by reducing thephosphofructokinase activity as compared to a non-modifiedmicroorganism” preferably refers to a microorganism in which theinactivation of the gene(s) encoding a phosphofructokinase or thereduction of the phosphofructokinase activity as compared to anon-modified microorganism is achieved by a genetic modification of themicroorganism which leads to said inactivation or reduction.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism that has an inactivatedEmbden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s)encoding a phosphofructokinase. The inactivation of the gene(s) encodinga phosphofructokinase in the context of the present invention means thatthe gene(s) coding for phosphofructokinase which are present in themicroorganism is (are) inactivated so that they are no longer expressedand/or do not lead to the synthesis of functional phosphofructokinase.Inactivation can be achieved by many different ways known in the art.The inactivation can, e.g., be achieved by the disruption of the gene(s)encoding the phosphofructokinase or by clean deletion of said gene(s)through the introduction of a selection marker. Alternatively, thepromoter of the gene(s) encoding the phosphofructokinase can be mutatedin a way that the gene is no longer transcribed into mRNA. Other ways toinactivate the gene(s) encoding the phosphofructokinase known in the artare: to express a polynucleotide encoding RNA having a nucleotidesequence complementary to the transcript of the phosphofructokinasegene(s) so that the mRNA can no longer be translated into a protein, toexpress a polynucleotide encoding RNA that suppresses the expression ofsaid gene(s) through RNAi effect; to express a polynucleotide encodingRNA having an activity of specifically cleaving a transcript of saidgene(s); or to express a polynucleotide encoding RNA that suppressesexpression of said gene(s) through co-suppression effect. Thesepolynucleotides can be incorporated into a vector, which can beintroduced into the microorganism by transformation to achieve theinactivation of the gene(s) encoding the phosphofructokinase.

The term “inactivation” in the context of the present inventionpreferably means complete inactivation, i.e. that the microorganism doesnot show phosphofructokinase activity. This means in particular that themicroorganism does not show phosphofructokinase activity independentfrom the used growth conditions. Preferably, “inactivation” means thatthe gene(s) encoding phosphofructokinase which are present in themicroorganism are genetically modified so as to prevent the expressionof the enzyme. This can be achieved, e.g. by deletion of the gene orparts thereof wherein the deletion of parts thereof prevents expressionof the enzyme, or by disruption of the gene either in the coding regionor in the promoter region wherein the disruption has the effect that noprotein is expressed or a dysfunctional protein is expressed.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism that has a diminishedEmbden-Meyerhof-Parnas pathway (EMPP) by reducing thephosphofructokinase activity as compared to a non-modifiedmicroorganism. Preferably, this reduction is achieved by a geneticmodification of the microorganism. This can be achieved e.g., by randommutagenesis or site-directed mutagenesis of the promoter and/or theenzyme and subsequent selection of promoters and/or enzymes having thedesired properties or by complementary nucleotide sequences or RNAieffect as described above. A detailed description of geneticmodification of microorganisms will be given further below.

In the context of the present invention, a “reduced activity” means thatthe expression and/or the activity of an enzyme, in particular of thephosphofructokinase, in the genetically modified microorganism is atleast 10%, preferably at least 20%, more preferably at least 30% or 50%,even more preferably at least 70% or 80% and particularly preferred atleast 90% or 100% lower than in the corresponding non-modifiedmicroorganism. Methods for measuring the level of expression of a givenprotein in a cell are well known to the person skilled in the art.Assays for measuring the reduced enzyme activity of aphosphofructokinase are known in the art.

In another embodiment the microorganism according to the presentinvention is a microorganism which does not possess aphosphofructokinase activity. This preferably means that such amicroorganism naturally does not possess a phosphofructokinase activity.This means that such a microorganism does naturally not contain in itsgenome a nucleotide sequence encoding an enzyme with phosphofructokinaseactivity. Examples for such microorganisms are Zymomonas mobilis (J. S.Suo et al., Nat. Biotechnol. 23:63 (2005)) and Ralstonia eutropha (C.Fleige et al., Appl. Microb. Cell Physiol. 91:769 (2011)).

The microorganism according to the present invention is furthercharacterised by having a diminished or inactivated oxidative branch ofthe pentose phosphate pathway (PPP) by inactivation of the gene(s)encoding a glucose-6-phosphate dehydrogenase or by reducing theglucose-6-phosphate dehydrogenase activity as compared to a non-modifiedmicroorganism or by not possessing glucose-6-phosphate dehydrogenaseactivity. Thus, the microorganism is either a microorganism whichnaturally has a PPP including glucose-6-phosphate dehydrogenase activitybut which has been modified, in particular genetically modified, so thatthe glucose-6-phosphate dehydrogenase activity is either completelyabolished or so that it is reduced compared to the correspondingnon-modified microorganism, or the microorganism is a microorganismwhich naturally does not possess a glucose-6-phosphate dehydrogenaseactivity.

Diminishing or inactivating the oxidative branch of the pentosephosphate pathway further increases the yield in acetyl-CoA sinceglucose-6-phosphate will no longer be drawn through the pentosephosphate cycle. All or almost all glucose-6-phosphate in themicroorganism will be converted into fructose-6-phosphate which willthen be further converted into acetyl-CoA.

The “glucose-6-phosphate dehydrogenase activity” means an enzymaticactivity that converts glucose-6-phosphate and NADP⁺ to6-phosphoglucono-δ-lactone and NADPH (EC 1.1.1.49). This enzymaticactivity can be measured by assays known in the art as, for example,described by Noltmann et al. (J. Biol. Chem. (1961) 236, 1225-1230).

The term “a microorganism which is characterised by having a diminishedor inactivated oxidative branch of the pentose phosphate pathway (PPP)by inactivation of the gene(s) encoding a glucose-6-phosphatedehydrogenase or by reducing the glucose-6-phosphate dehydrogenaseactivity as compared to a non-modified microorganism” preferably refersto a microorganism in which the inactivation of the gene(s) encoding aglucose-6-phosphate dehydrogenase or the reduction of theglucose-6-phosphate dehydrogenase activity as compared to a non-modifiedmicroorganism is achieved by a genetic modification of the microorganismwhich leads to said inactivation or reduction.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism that has an inactivatedoxidative branch of the pentose phosphate pathway (PPP) by inactivationof the gene(s) encoding a glucose-6-phosphate dehydrogenase. Theinactivation of the gene(s) encoding a glucose-6-phosphate dehydrogenasein the context of the present invention means that the gene(s) codingfor glucose-6-phosphate dehydrogenase which is (are) present in themicroorganism is (are) inactivated so that they are no longer expressedand/or do not lead to the synthesis of functional glucose-6-phosphatedehydrogenase. Inactivation can be achieved by many different ways knownin the art. The inactivation can, e.g., be achieved by the disruption ofthe gene(s) encoding the glucose-6-phosphate dehydrogenase or by cleandeletion of said gene(s) through the introduction of a selection marker.Alternatively, the promoter of the gene(s) encoding theglucose-6-phosphate dehydrogenase can be mutated in a way that thegene(s) is/are no longer transcribed into mRNA. Other ways to inactivatethe gene(s) encoding the phosphofructokinase known in the art are: toexpress a polynucleotide encoding RNA having a nucleotide sequencecomplementary to the transcript of the glucose-6-phosphate dehydrogenasegene(s) so that the mRNA can no longer be translated into a protein, toexpress a polynucleotide encoding RNA that suppresses the expression ofsaid gene(s) through RNAi effect; to express a polynucleotide encodingRNA having an activity of specifically cleaving a transcript of saidgene(s); or to express a polynucleotide encoding RNA that suppressesexpression of said gene(s) through co-suppression effect. Thesepolynucleotides can be incorporated into a vector, which can beintroduced into the microorganism by transformation to achieve theinactivation of the gene(s) encoding the glucose-6-phosphatedehydrogenase.

The term “inactivation” in the context of the present inventionpreferably means complete inactivation, i.e. that the microorganism doesnot show glucose-6-phosphate dehydrogenase activity. This means inparticular that the microorganism does not show glucose-6-phosphatedehydrogenase activity independent from the used growth conditions.

Preferably, “inactivation” means that the gene(s) encodingglucose-6-phosphate dehydrogenase which are present in the microorganismare genetically modified so as to prevent the expression of the enzyme.This can be achieved, e.g. by deletion of the gene or parts thereofwherein the deletion of parts thereof prevents expression of the enzyme,or by disruption of the gene either in the coding region or in thepromoter region wherein the disruption has the effect that no protein isexpressed or a dysfunctional protein is expressed.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism that has a diminished oxidativebranch of the pentose phosphate pathway (PPP) by reducing theglucose-6-phosphate dehydrogenase activity as compared to a non-modifiedmicroorganism. Preferably, this reduction is achieved by a geneticmodification of the microorganism. This can be achieved e.g., by randommutagenesis or site-directed mutagenesis of the promoter and/or theenzyme and subsequent selection of promoters and/or enzymes having thedesired properties or by complementary nucleotide sequences or RNAieffect as described above. A detailed description of geneticmodification of microorganisms will be given further below.

In the context of the present invention, a “reduced activity” means thatthe expression and/or the activity of an enzyme, in particular of theglucose-6-phosphate dehydrogenase, in the genetically modifiedmicroorganism is at least 10%, preferably at least 20%, more preferablyat least 30% or 50%, even more preferably at least 70% or 80% andparticularly preferred at least 90% or 100% lower than in thecorresponding non-modified microorganism. Methods for measuring thelevel of expression of a given protein in a cell are well known to theperson skilled in the art. Assays for measuring the reduced enzymeactivity of a glucose-6-phosphate dehydrogenase are known in the art.

In another embodiment the microorganism according to the presentinvention is a microorganism which does not possess aglucose-6-phosphate dehydrogenase activity. This preferably means thatsuch a microorganism naturally does not possess a glucose-6-phosphatedehydrogenase activity. This means that such a microorganism doesnaturally not contain in its genome a nucleotide sequence encoding anenzyme with glucose-6-phosphate dehydrogenase activity. Examples forsuch microorganisms are Acinetobacter baylyi (Barbe et al., Nucl. AcidsRes. 32 (2004), 5766-5779), archae of the hyperthermophilic phylum suchas Sulfolobus solfataricus (Nunn et al., J. Biol. Chem. 285 (2010),33701-33709), Thermoproteus tenax, Thermoplasma acidophilum andPicrophilus torridus (Reher and Schönheit, FEBS Lett. 580 (2006),1198-1204).

In a further embodiment, the microorganism according to the presentinvention is further characterised by having fructose-1,6-bisphosphatephosphatase activity, preferably when grown on glucose.Fructose-1,6-bisphosphate phosphatase is an enzyme participating in thegluconeogenesis hydrolyzing fructose-1,6-bisphosphate intofructose-6-phosphate and free phosphate. However, in basically allorganisms in the presence of glucose, fructose-1,6-bisphosphatephosphatase activity is repressed and glucose is processed through EMPP(glycolysis). The microorganism of the present invention, which hasphosphoketolase activity and which does not possess phosphofructokinaseactivity or in which phosphofructokinase activity is reduced or whosegene encoding the phosphofructokinase is inactivated, the yield ofacetyl-CoA by conversion of fructose-6-phosphate with thephosphoketolase (EC 4.1.2.9 or EC 4.1.2.22) can be enhanced by ensuringthe presence of fructose-1,6-bisphosphate phosphatase activity, forexample by derepression of the fructose-1,6-bisphosphate phosphatase inthe presence of glucose. The presence of fructose-1,6-bisphosphatephosphatase activity results in the recycling offructose-1,6-bisphosphate produced by fructose-1,6-bisphosphate aldolaseinto fructose-6-phosphate which can then again be converted via thephosphoketolase pathway to acetyl-CoA. Indeed, the product acetylphosphate of phosphoketolase interconverts into acetyl-CoA through theaction of the enzyme phosphate acetyltransferase EC 2.3.1.8. Thus, therecombinant microorganism of the present invention is capable ofproducing acetyl-CoA from glucose at a stoichiometry approaching 3:1.The sum of the reactions is given in equation 2:glucose+ATP+3CoA→3acetyl-CoA+ADP+H₃PO₄+2H₂O   (Equation 2)

The term “fructose-1,6-bisphosphate phosphatase activity” means anenzymatic activity that converts fructose-1,6-bisphosphate and H₂O tofructose-6-phosphate and phosphate (EC 3.1.3.11). This enzymaticactivity can be measured by assays known in the art as, for example,described by Hines et al. (J. Biol. Chem. (2007) 282, 11696-11704). Theterms “fructose-1,6-bisphosphate phosphatase activity” and“fructose-1,6-bisphosphate phosphatase” also cover enzymes which arebifunctional in the sense that they show a fructose-1-6-bisphosphatealdolase/phosphatase activity. Such bifunctional enzymes are expressedin most archaeal and deeply branching bacterial lineages and, in mostcases, are heat-stable. Such enzymes are, for example, reported forThermococcus kodakaraensis, Sulfolobus tokodaii, Ignicoccus hospitalis,Cenarchaeum symbiosum, Sulfolobus solfataricas, Thermus thermophilus,Thermoproteus neutrophilus, Moorella thermoacetica and many others (see,e.g., Say and Fuchs (Nature 464 (2010), 1077); Fushinobu et al. (Nature478 (2011), 538; Du et al. (Nature 478 (2011), 534).

The term “fructose-1,6-bisphosphate phosphatase activity when grown onglucose” means that the microorganism expresses an enzyme withfructose-1,6-bisphosphate phosphatase activity when the microorganism isgrown on glucose. “Grown on glucose” means that the microorganism isgrown in a medium which contains inter alia glucose as carbon source.Preferably, this term means that the microorganism is grown in a mediumcontaining glucose as sole carbon source.

In the context of the present invention, a microorganism which hasfructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, can, for example, be a microorganism which naturally hasfructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, or a microorganism that does not naturally havefructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, and that has been genetically modified to express afructose-1,6-bisphosphate phosphatase, in particular when grown onglucose. It may also be a microorganism which naturally hasfructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, and which has been genetically modified, e.g. transformedwith a nucleic acid, e.g. a vector, encoding a fructose-1,6-bisphosphatephosphatase in order to increase the phosphoketolase activity in saidmicroorganism.

Microorganisms that inherently, i.e. naturally, havefructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, are known in the art and any of them can be used in thecontext of the present invention.

It is also possible in the context of the present invention that themicroorganism is a microorganism which naturally does not havefructose-1,6-bisphosphate phosphatase activity, in particular when grownon glucose, but which is genetically modified so as to be able toexpress a fructose-1,6-bisphosphate phosphatase, in particular, whengrown on glucose. This can be achieved, e.g., by mutating the promoterof the gene encoding the fructose-1,6-bisphosphate phosphatase in a waythat the gene is no longer repressed when the microorganism is grown onglucose or the promoter can be replaced by another promoter e.g. aconstitutive promoter which is not regulated when the microorganism isgrown on glucose.

Similarly, the microorganism may also be a microorganism which naturallyhas fructose-1,6-bisphosphate phosphatase activity, in particular whengrown on glucose, but which is genetically modified so as toenhance/increase the fructose-1,6-bisphosphate phosphatase activity, inparticular when grown on glucose, e.g. by the introduction of anexogenous nucleotide sequence encoding a fructose-1,6-bisphosphatephosphatase.

The genetic modification of microorganisms to express an enzyme ofinterest will be described in detail below.

The fructose-1,6-bisphosphate phosphatase according to the invention canbe a naturally occurring fructose-1,6-bisphosphate phosphatase or it canbe a fructose-1,6-bisphosphate phosphatase which is derived from anaturally occurring fructose-1,6-bisphosphate phosphatase, e.g. by theintroduction of mutations or other alterations which, e.g., alter orimprove the enzymatic activity, the stability, etc. Methods formodifying and/or improving the desired enzymatic activities of proteinsare well-known to the person skilled in the art and have been describedabove. The resulting fructose-1,6-bisphosphate phosphatase variants arethen tested for their properties, e.g. enzymatic activity or regulation.Assays for measuring the enzyme activity of a fructose-1,6-bisphosphatephosphatase are known in the art. In one embodiment thefructose-1,6-bisphosphate phosphatase is an enzyme which is notregulated by feed-back inhibition.

In a preferred embodiment, the recombinant microorganism has beengenetically modified to have an increased fructose-1,6-bisphosphatephosphatase activity. This can be achieved e.g. by transforming themicroorganism with a nucleic acid encoding a fructose-1,6-bisphosphatephosphatase. A detailed description of genetic modification ofmicroorganisms will be given further below.

In the context of the present invention, an “increased activity” meansthat the expression and/or the activity of an enzyme, in particular ofthe fructose-1,6-bisphosphate phosphatase, in the genetically modifiedmicroorganism when grown on glucose is at least 10%, preferably at least20%, more preferably at least 30% or 50%, even more preferably at least70% or 80% and particularly preferred at least 90% or 100% higher thanin the corresponding non-modified microorganism when grown on glucose.In even more preferred embodiments the increase in expression and/oractivity may be at least 150%, at least 200% or at least 500%. Inparticularly preferred embodiments the expression is at least 10-fold,more preferably at least 100-fold and even more preferred at least1000-fold higher than in the corresponding non-modified microorganism inparticular when grown on glucose.

Methods for measuring the level of expression of a given protein in acell are well known to the person skilled in the art. In one embodiment,the measurement of the level of expression is done by measuring theamount of the corresponding protein. Corresponding methods are wellknown to the person skilled in the art and include Western Blot, ELISAetc. In another embodiment the measurement of the level of expression isdone by measuring the amount of the corresponding RNA. Correspondingmethods are well known to the person skilled in the art and include,e.g., Northern Blot.

Methods for measuring the enzymatic activity of thefructose-1,6-bisphosphate are known in the art.

In another embodiment, the microorganism according to the presentinvention is further characterised in that the EMPP is furtherdiminished or inactivated by inactivation of the gene(s) encoding theglyceraldehyde 3-phosphate dehydrogenase or by reducing theglyceraldehyde 3-phosphate dehydrogenase activity as compared to anon-modified microorganism. Further diminishing the EMPP at a stepfurther downstream by diminishing or inactivating the glyceraldehyde3-phosphate dehydrogenase ensures that none or almost noneglyceraldehyde 3-phosphate that may be produced in the microorganismwill be processed via the glycolysis to acetyl-CoA whereby one carbonatom would be lost by the release of CO₂ in the last step catalysed bythe pyruvate dehydrogenase. Therefore, blocking the EMPP by diminishingor inactivating the glyceraldehyde 3-phosphate dehydrogenase activityfurther ensures that the overall flux is directed towards thephosphoketolase.

The “glyceraldehyde 3-phosphate dehydrogenase activity” means anenzymatic activity that converts glyceraldehyde 3-phosphate, phosphateand NAD⁺ to 3-phospho-D-glyceroyl phosphate and NADH+H⁺ (EC 1.2.1.12).This activity can be measured by assays known in the art as, forexample, described by D'Alessio et al. (J. Biol. Chem. (1971) 246,4326-4333).

The term “a microorganism which is characterised by having a furtherdiminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP) byinactivation of the gene(s) encoding a glyceraldehyde 3-phosphatedehydrogenase or by reducing the glyceraldehyde 3-phosphatedehydrogenase activity as compared to a non-modified microorganism”preferably refers to a microorganism in which the inactivation of thegene(s) encoding a glyceraldehyde 3-phosphate dehydrogenase or thereduction of the glyceraldehyde 3-phosphate dehydrogenase activity ascompared to a non-modified microorganism is achieved by a geneticmodification of the microorganism which leads to said inactivation orreduction.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism in which the EMPP is furtherdiminished or inactivated by inactivation of the gene(s) encoding theglyceraldehyde 3-phosphate dehydrogenase or by reducing theglyceraldehyde 3-phosphate dehydrogenase activity as compared to anon-modified microorganism. The inactivation of the gene(s) encoding aglyceraldehyde 3-phosphate dehydrogenase in the context of the presentinvention means that the gene(s) coding for glyceraldehyde 3-phosphatedehydrogenase which is (are) present in the microorganism is (are)inactivated so that they are no longer expressed and/or do not lead tothe synthesis of functional glyceraldehyde 3-phosphate dehydrogenase.Inactivation can be achieved by many different ways known in the art.The inactivation can, e.g., be achieved by the disruption of the gene(s)encoding the glyceraldehyde 3-phosphate dehydrogenase or by cleandeletion of said gene(s) through the introduction of a selection marker.Alternatively, the promoter of the gene encoding the glyceraldehyde3-phosphate dehydrogenase can be mutated in a way that the gene(s)is/are no longer transcribed into mRNA. Other ways to inactivate thegene(s) encoding the glyceraldehyde 3-phosphate dehydrogenase known inthe art are: to express a polynucleotide encoding RNA having anucleotide sequence complementary to the transcript of theglyceraldehyde 3-phosphate dehydrogenase gene(s) so that the mRNA can nolonger be translated into a protein, to express a polynucleotideencoding RNA that suppresses the expression of said gene(s) through RNAieffect; to express a polynucleotide encoding RNA having an activity ofspecifically cleaving a transcript of said gene(s); or to express apolynucleotide encoding RNA that suppresses expression of said gene(s)through co-suppression effect. These polynucleotides can be incorporatedinto a vector, which can be introduced into the microorganism bytransformation to achieve the inactivation of the gene(s) encoding theglyceraldehyde 3-phosphate dehydrogenase.

The term “inactivation” in the context of the present inventionpreferably means complete inactivation, i.e. that the microorganism doesnot show glyceraldehyde 3-phosphate dehydrogenase activity. This meansin particular that the microorganism does not show glyceraldehyde3-phosphate dehydrogenase activity independent from the used growthconditions.

Preferably, “inactivation” means that the gene(s) encodingglyceraldehyde 3-phosphate dehydrogenase which are present in themicroorganism are genetically modified so as to prevent the expressionof the enzyme. This can be achieved, e.g. by deletion of the gene orparts thereof wherein the deletion of parts thereof prevents expressionof the enzyme, or by disruption of the gene either in the coding regionor in the promoter region wherein the disruption has the effect that noprotein is expressed or a dysfunctional protein is expressed.

In a preferred embodiment, the recombinant microorganism of the presentinvention is a recombinant microorganism that has a diminished EMPP byreducing the glyceraldehyde 3-phosphate dehydrogenase activity ascompared to a non-modified microorganism. Preferably, this reduction isachieved by a genetic modification of the microorganism. This can beachieved e.g., by random mutagenesis or site-directed mutagenesis of thepromoter and/or the enzyme and subsequent selection of promoters and/orenzymes having the desired properties or by complementary nucleotidesequences or RNAi effect as described above. A detailed description ofgenetic modification of microorganisms will be given further below.

In the context of the present invention, a “reduced activity” means thatthe expression and/or the activity of an enzyme, in particular of theglyceraldehyde 3-phosphate dehydrogenase, in the genetically modifiedmicroorganism is at least 10%, preferably at least 20%, more preferablyat least 30% or 50%, even more preferably at least 70% or 80% andparticularly preferred at least 90% or 100% lower than in thecorresponding non-modified microorganism. Methods for measuring thelevel of expression of a given protein in a cell are well known to theperson skilled in the art. Assays for measuring the reduced enzymeactivity of a glyceraldehyde 3-phosphate dehydrogenase are known in theart.

The term “microorganism” in the context of the present invention refersto bacteria, as well as to fungi, such as yeasts, and also to algae andarchaea. In one preferred embodiment, the microorganism is a bacterium.In principle any bacterium can be used. Preferred bacteria to beemployed in the process according to the invention are bacteria of thegenus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas orEscherichia. In a particularly preferred embodiment the bacteriumbelongs to the genus Escherichia and even more preferred to the speciesEscherichia coli. In another preferred embodiment the bacterium belongsto the species Pseudomonas putida or to the species Zymomonas mobilis orto the species Corynebacterium glutamicum.

In another preferred embodiment the microorganism is a fungus, morepreferably a fungus of the genus Saccharomyces, Schizosaccharomyces,Aspergillus, Trichoderma, Kluyveromyces or Pichia and even morepreferably of the species Saccharomyces cerevisiae, Schizosaccharomycespombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus,Kluyveromyces lactis, Pichia pastoris, Pichia torula or Pichia utilis.

In a more preferred embodiment, where the recombinant microorganism is abacterium, the gene(s) encoding the PEP-dependent PTS transporter havebeen inactivated. In the context of the present invention, inactivationmeans that the gene(s) coding for PEP-dependent PTS transporter which is(are) present in the microorganism is (are) inactivated so that they areno longer expressed and/or do not lead to the synthesis of functionalPEP-dependent PTS transporter. The inactivation of the gene(s) encodingthe PEP-dependent PTS transporter should be such that the bacteria areno longer capable of transporting glucose via the PEP-dependent PTStransporter.

PEP-dependent PTS transporter (e.g. from E. coli, B. subtilis) are knownin the art. An example for inactivation of the PEP-dependent PTStransporter is shown in the Example section below.

Inactivation can be achieved by many different ways known in the art.The inactivation can, e.g., be achieved by the disruption of the gene(s)encoding the PEP-dependent PTS transporter or by clean deletion of saidgene(s) through the introduction of a selection marker. Alternatively,the promoter of the gene(s) encoding the PEP-dependent PTS transportercan be mutated in a way that the gene(s) is (are) no longer transcribedinto mRNA. Other ways to inactivate the gene(s) encoding thePEP-dependent PTS transporter known in the art are: to express apolynucleotide encoding RNA having a nucleotide sequence complementaryto the transcript of the PEP-dependent PTS transporter gene(s) so thatthe mRNA can no longer be translated into a protein, to express apolynucleotide encoding RNA that suppresses the expression of saidgene(s) through RNAi effect; to express a polynucleotide encoding RNAhaving an activity of specifically cleaving a transcript of saidgene(s); or to express a polynucleotide encoding RNA that suppressesexpression of said gene(s) through co-suppression effect. Thesepolynucleotides can be incorporated into a vector, which can beintroduced into the microorganism by transformation to achieve theinactivation of the gene(s) encoding the PEP-dependent PTS transporter.

The term “recombinant” means that the microorganism of the presentinvention is genetically modified so as to contain a nucleic acidmolecule encoding an enzyme as defined above as compared to a wild-typeor non-modified microorganism or so that a gene encoding an enzyme asdefined above has been deleted as compared to a wild-type ornon-modified microorganism.

A nucleic acid molecule encoding an enzyme as defined above can be usedalone or as part of a vector.

The nucleic acid molecules can further comprise expression controlsequences operably linked to the polynucleotide comprised in the nucleicacid molecule. The term “operatively linked” or “operably linked”, asused throughout the present description, refers to a linkage between oneor more expression control sequences and the coding region in thepolynucleotide to be expressed in such a way that expression is achievedunder conditions compatible with the expression control sequence.

Expression comprises transcription of the heterologous DNA sequence,preferably into a translatable mRNA. Regulatory elements ensuringexpression in fungi as well as in bacteria, are well known to thoseskilled in the art. They encompass promoters, enhancers, terminationsignals, targeting signals and the like. Examples are given furtherbelow in connection with explanations concerning vectors.

Promoters for use in connection with the nucleic acid molecule may behomologous or heterologous with regard to its origin and/or with regardto the gene to be expressed. Suitable promoters are for instancepromoters which lend themselves to constitutive expression. However,promoters which are only activated at a point in time determined byexternal influences can also be used. Artificial and/or chemicallyinducible promoters may be used in this context.

The vectors can further comprise expression control sequences operablylinked to said polynucleotides contained in the vectors. Theseexpression control sequences may be suited to ensure transcription andsynthesis of a translatable RNA in bacteria or fungi.

In addition, it is possible to insert different mutations into thepolynucleotides by methods usual in molecular biology (see for instanceSambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSHPress, Cold Spring Harbor, N.Y., USA), leading to the synthesis ofpolypeptides possibly having modified biological properties. Theintroduction of point mutations is conceivable at positions at which amodification of the amino acid sequence for instance influences thebiological activity or the regulation of the polypeptide.

Moreover, mutants possessing a modified substrate or product specificitycan be prepared. Preferably, such mutants show an increased activity.Alternatively, mutants can be prepared the catalytic activity of whichis abolished without losing substrate binding activity.

Furthermore, the introduction of mutations into the polynucleotidesencoding an enzyme as defined above allows the gene expression rateand/or the activity of the enzymes encoded by said polynucleotides to bereduced or increased.

For genetically modifying bacteria or fungi, the polynucleotidesencoding an enzyme as defined above or parts of these molecules can beintroduced into plasmids which permit mutagenesis or sequencemodification by recombination of DNA sequences. Standard methods (seeSambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSHPress, Cold Spring Harbor, N.Y., USA) allow base exchanges to beperformed or natural or synthetic sequences to be added. DNA fragmentscan be connected to each other by applying adapters and linkers to thefragments. Moreover, engineering measures which provide suitablerestriction sites or remove surplus DNA or restriction sites can beused. In those cases, in which insertions, deletions or substitutionsare possible, in vitro mutagenesis, “primer repair”, restriction orligation can be used. In general, a sequence analysis, restrictionanalysis and other methods of biochemistry and molecular biology arecarried out as analysis methods.

Thus, in accordance with the present invention a recombinantmicroorganism can be produced by genetically modifying fungi or bacteriacomprising introducing the above-described polynucleotides, nucleic acidmolecules or vectors into a fungus or bacterium.

The invention relates to recombinant microorganisms, in particularbacteria and fungi, genetically modified with the above-describedpolynucleotides, nucleic acid molecules or vectors or obtainable by theabove-mentioned method for producing genetically modified bacteria orfungi, and to cells derived from such transformed bacteria or fungi andcontaining a polynucleotide, nucleic acid molecule or vector as definedabove. In a preferred embodiment the host cell is genetically modifiedin such a way that it contains the polynucleotide stably integrated intothe genome.

The polynucleotide is expressed so as to lead to the production of apolypeptide having any of the activities described above. An overview ofdifferent expression systems is for instance contained in Methods inEnzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology153 (1987), 516-544) and in Sawers et al. (Applied Microbiology andBiotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion inBiotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12(1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75(1997), 427-440). An overview of yeast expression systems is forinstance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995),261-279), Bussineau et al. (Developments in Biological Standardization83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992),79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496),Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) andBuckholz (Bio/Technology 9 (1991), 1067-1072).

Expression vectors have been widely described in the literature. As arule, they contain not only a selection marker gene and areplication-origin ensuring replication in the host selected, but also abacterial or viral promoter, and in most cases a termination signal fortranscription. Between the promoter and the termination signal there isin general at least one restriction site or a polylinker which enablesthe insertion of a coding DNA sequence. The DNA sequence naturallycontrolling the transcription of the corresponding gene can be used asthe promoter sequence, if it is active in the selected host organism.However, this sequence can also be exchanged for other promotersequences. It is possible to use promoters ensuring constitutiveexpression of the gene and inducible promoters which permit a deliberatecontrol of the expression of the gene. Bacterial and viral promotersequences possessing these properties are described in detail in theliterature. Regulatory sequences for the expression in microorganisms(for instance E. coli, S. cerevisiae) are sufficiently described in theliterature. Promoters permitting a particularly high expression of adownstream sequence are for instance the T7 promoter (Studier et al.,Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5(DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structureand Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc.Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Gene 42(1986), 97-100). Inducible promoters are preferably used for thesynthesis of polypeptides. These promoters often lead to higherpolypeptide yields than do constitutive promoters. In order to obtain anoptimum amount of polypeptide, a two-stage process is often used. First,the host cells are cultured under optimum conditions up to a relativelyhigh cell density. In the second step, transcription is induceddepending on the type of promoter used. In this regard, a tac promoteris particularly suitable which can be induced by lactose or IPTG(=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad.Sci. USA 80 (1983), 21-25). Termination signals for transcription arealso described in the literature.

The transformation of the host cell with a polynucleotide or vectoraccording to the invention can be carried out by standard methods, asfor instance described in Sambrook and Russell (2001), MolecularCloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA;Methods in Yeast Genetics, A Laboratory Course Manual, Cold SpringHarbor Laboratory Press, 1990. The host cell is cultured in nutrientmedia meeting the requirements of the particular host cell used, inparticular in respect of the pH value, temperature, salt concentration,aeration, antibiotics, vitamins, trace elements etc.

In another aspect of the present invention, the recombinantmicroorganism is further characterized in that it is capable ofconverting acetyl-CoA into acetone. Methods for providing such arecombinant microorganism are for instance disclosed in EP 2 295 593.The term “which is capable of converting acetyl-CoA into acetone” in thecontext of the present invention means that the organism/microorganismhas the capacity to produce acetone within the cell due to the presenceof enzymes providing enzymatic activities allowing the production ofacetone from acetyl-CoA.

Acetone is produced by certain microorganisms, such as Clostridiumacetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum,Bacillus polymyxa and Pseudomonas putida. The synthesis of acetone isbest characterized in Clostridium acetobutylicum. It starts out with areaction (reaction step 1) in which two molecules of acetyl-CoA arecondensed into acetoacetyl-CoA. This reaction is catalyzed by acetyl-CoAacetyltransferase (EC 2.3.1.9). Acetoacetyl-CoA is then converted intoacetoacetate by a reaction with acetic acid or butyric acid resultingalso in the production of acetyl-CoA or butyryl-CoA (reaction step 2).This reaction is catalyzed e.g. by acetoacetylCoA transferase (EC2.8.3.8). AcetoacetylCoA transferase is known from various organisms,e.g. from E. coli in which it is encoded by the atoAD gene or fromClostridium acetobutylicum in which it is encoded by the ctfAB gene.However, also other enzymes can catalyze this reaction, e.g. 3-oxoacidCoA transferase (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5).

Finally, acetoacetate is converted into acetone by a decarboxylationstep (reaction step 3) catalyzed by acetoacetate decarboxylase (EC4.1.1.4).

The above described reaction steps 1 and 2 and the enzymes catalyzingthem are not characteristic for the acetone synthesis and can be foundin various organism. In contrast, reaction step 3 which is catalyzed byacetoacetate decarboxylase (EC 4.1.1.4) is only found in those organismswhich are capable of producing acetone.

In a preferred embodiment the recombinant microorganism of the presentinvention is a microorganism, which naturally has the capacity toproduce acetone. Thus, preferably the microorganism belongs to the genusClostridium, Bacillus or Pseudomonas, more preferably to the speciesClostridium acetobutylicum, Clostridium beijerinckii, Clostridiumcellulolyticum, Bacillus polymyxa or Pseudomonas putida.

In another preferred embodiment, the recombinant microorganism of thepresent invention is a microorganism, derived from anorganism/microorganism which naturally does not produce acetone butwhich has been genetically modified so as to produce acetone, i.e. byintroducing the gene(s) necessary for allowing the production of acetonein the microorganism. In principle any microorganism can be geneticallymodified in this way. The enzymes responsible for the synthesis ofacetone have been described above. Genes encoding corresponding enzymesare known in the art and can be used to genetically modify a givenmicroorganism so as to produce acetone. As described above, the reactionsteps 1 and 2 of the acetone synthesis occur naturally in mostorganisms. However, reaction step 3 is characteristic and crucial foracetone synthesis. Thus, in a preferred embodiment, a geneticallymodified microorganism derived from a microorganism which naturally doesnot produce acetone is modified so as to contain a nucleotide sequenceencoding an enzyme catalyzing the conversion of acetoacetate intoacetone by decarboxylation, e.g. an acetoacetate decarboxylase (EC4.1.1.4). Nucleotide sequences from several organisms encoding thisenzyme are known in the art, e.g. the adc gene from Clostridiumacetobutylicum (Uniprot accession numbers P23670 and P23673),Clostridium beijerinckii (Clostridium MP; Q9RPK1), Clostridiumpasteurianum (Uniprot accession number P81336), Bradyrhizobium sp.(strain BTAi1/ATCC BAA-1182; Uniprot accession number A5EBU7),Burkholderia mallei (ATCC 10399 A9LBS0), Burkholderia mallei (Uniprotaccession number A3MAE3), Burkholderia mallei FMH A5XJB2, Burkholderiacenocepacia (Uniprot accession number A0B471), Burkholderia ambifaria(Uniprot accession number Q0b5P1), Burkholderia phytofirmans (Uniprotaccession number B2T319), Burkholderia spec. (Uniprot accession numberQ38ZU0), Clostridium botulinum (Uniprot accession number B2TLN8),Ralstonia pickettii (Uniprot accession number B2UIG7), Streptomycesnogalater (Uniprot accession number Q9EYI7), Streptomyces avermitilis(Uniprot accession number Q82NF4), Legionella pneumophila (Uniprotaccession number Q5ZXQ9), Lactobacillus salivarius (Uniprot accessionnumber Q1WVG5), Rhodococcus spec. (Uniprot accession number QOS7W4),Lactobacillus plantarum (Uniprot accession number Q890G0), Rhizobiumleguminosarum (Uniprot accession number Q1M911), Lactobacillus casei(Uniprot accession number Q03B66), Francisella tularensis (Uniprotaccession number QOBLC9), Saccharopolyspora erythreae (Uniprot accessionnumber A4FKR9), Korarchaeum cryptofilum (Uniprot accession numberB1L3N6), Bacillus amyloliquefaciens (Uniprot accession number A7Z8K8),Cochliobolus heterostrophus (Uniprot accession number Q8NJQ3),Sulfolobus islandicus (Uniprot accession number C3ML22) and Francisellatularensis subsp. holarctica (strain OSU18).

More preferably, the microorganism is genetically modified so as to betransformed with a nucleic acid molecule encoding an enzyme capable ofcatalyzing the above mentioned reaction step 2 of the acetone synthesis,i.e. the conversion of acetoacetyl CoA into acetoacetate.

Even more preferably, the microorganism is genetically modified so as tobe transformed with a nucleic acid molecule encoding an enzyme capableof catalyzing the above mentioned reaction step 1 of the acetonesynthesis, i.e. the condensation of two molecules of acetyl CoA intoacetoacetatyl CoA.

In a particularly preferred embodiment the microorganism is geneticallymodified so as to be transformed with a nucleic acid molecule encodingan enzyme capable of catalyzing the above mentioned reaction step 1 ofthe acetone synthesis and with a nucleic acid molecule encoding anenzyme capable of catalyzing the above mentioned reaction step 2 of theacetone synthesis or with a nucleic acid molecule encoding an enzymecapable of catalyzing the above mentioned reaction step 1 of the acetonesynthesis and with a nucleic acid molecule encoding an enzyme capable ofcatalyzing the above mentioned reaction step 3 of the acetone synthesisor with a nucleic acid molecule encoding an enzyme capable of catalyzingthe above mentioned reaction step 2 of the acetone synthesis and with anucleic acid molecule encoding an enzyme capable of catalyzing the abovementioned reaction step 3 of the acetone synthesis or with a nucleicacid molecule encoding an enzyme capable of catalyzing the abovementioned reaction step 1 of the acetone synthesis and with a nucleicacid molecule encoding an enzyme capable of catalyzing the abovementioned reaction step 2 of the acetone synthesis and with a nucleicacid molecule encoding an enzyme capable of catalyzing the abovementioned reaction step 3 of the acetone synthesis.

Methods for preparing the above mentioned genetically modifiedmicroorganisms are well known in the art. Thus, generally, themicroorganism is transformed with a DNA construct allowing expression ofthe respective enzyme in the microorganism. Such a construct normallycomprises the coding sequence in question linked to regulatory sequencesallowing transcription and translation in the respective host cell, e.g.a promoter and/enhancer and/or transcription terminator and/or ribosomebinding sites etc. The prior art already describes microorganisms whichhave been genetically modified so as to be able to produce acetone. Inparticular genes from, e.g., Clostridium acetobutylicum have beenintroduced into E. coli thereby allowing the synthesis of acetone in E.coli, a bacterium which naturally does not produce acetone (Bermejo etal., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl.Environ. Microbiol. 73 (2007), 7814-7818). In particular Hanai et al.(loc. cit.) shows that it is sufficient to introduce a nucleic acidsequence encoding an acetoacetate decarboxylase (such as that fromClostridium acetobutylicum) in order to achieve acetone production in E.coli indicating that the endogenous enzymes in E. coli catalyzing theabove-mentioned reaction steps 1 and 2 (i.e. the expression products ofthe E. coli atoB and atoAD genes) are sufficient to provide substratefor the acetone production.

In another aspect of the present invention, the recombinantmicroorganism is further characterized in that it is capable ofconverting acetyl-CoA into acetone and converting acetone intoisobutene. Methods for providing such a recombinant microorganism arefor instance disclosed in EP-A 2 295 593 (EP 09 17 0312), WO 2010/001078and EP 10 18 8001.

In another aspect of the present invention, the recombinantmicroorganism is characterized in that it is capable of convertingacetyl-CoA into acetone and converting acetone into propene. Methods forproviding such a recombinant microorganism are for instance disclosed inHanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818.

One skilled in the art would recognize that further geneticmodifications to the microorganisms of the present invention could leadto improvements in the efficacy by which the microorganisms of thepresent invention convert feedstock to product. For example, naturalmicroorganisms commonly produce products such as formate, acetate,lactate, succinate, ethanol, glycerol, 2,3-butanediol, methylglyoxal andhydrogen; all of which would be deleterious to the production of, e.g.,acetone, isobutene or propene from sugars. Elimination or substantialreduction of such unwanted by-products may be achieved by elimination orreduction of key enzymes activities leading their production. Suchactivities include, but are not limited to, the group consisting of:

-   -   acetyl-CoA+formate=CoA+pyruvate (for example, catalyzed by        formate C-acetyltransferase, also known as pyruvate        formate-lyase (EC 2.3.1.54); for E. coli-pflB, NOBI-GeneID:        945514);    -   ATP+acetate=ADP+acetyl phosphate (for example, catalyzed by        acetate kinase (EC 2.7.2.1); for E. coli-ackA, NOBI-GeneID:        946775);    -   (R)-lactate+NAD⁺=pyruvate+NADH+H⁺ (for example, catalyzed by        L-lactate dehydrogenase (EC 1.1.1.28); for E. coli-IdhA,        NOBI-GeneID: 946315);    -   phosphate+oxaloacetate=phosphoenolpyruvate+HCO₃ ⁻ (for example,        catalyzed by phosphoenolpyruvate carboxylase (EC 4.1.1.31);        for E. coli-ppc, NOBI-GeneID: 948457);    -   ATP+oxaloacetate=ADP+phosphoenolpyruvate+CO₂ (for example,        catalyzed by phosphoenolpyruvate carboxykinase (ATP) (EC        4.1.1.49); for E. coli-pck, NOBI-GeneID: 945667);    -   succinate+acceptor=fumarate+reduced acceptor (for example,        catalyzed by succinate dehydrogenase (EC 1.3.99.1); for E.        coli—comprising frdA and frdB, NOBI-GeneID: 948667 and 948666,        respectively);    -   a 2-oxo carboxylate (e.g. pyruvate)=an aldehyde (e.g.        acetaldehyde+CO₂ (for example, catalyzed by pyruvate        decarboxylase (EC 4.1.1.1));    -   acetaldehyde+CoA+NAD⁺=acetyl-CoA+NADH+H⁺ (for example, catalyzed        by acetaldehyde dehydrogenase (acetylating) (EC 1.2.1.10);        for E. coli-adhE, NCBI-GeneID: 945837);    -   sn-glycerol 3-phosphate+NAD(P)⁺=glycerone phosphate+NAD(P)H+H⁺        (for example, catalyzed by glycerol-3-phosphate dehydrogenase        [NAD(P)⁺] (EC 1.1.1.94); for E. coli—gpsA, NCBI-GeneID: 948125);    -   2 pyruvate=2-acetolactate+CO₂ (for example, catalyzed by        acetolactate synthase (EC 2.2.1.6); for E. coli-ilvH and ilvI,        NCBI-GeneID: 947267 and 948793, respectively);    -   glycerone phosphate=methylglyoxal+phosphate (for example,        catalyzed by methylglyoxal synthase (EC 4.2.3.3); for E.        coli-mgsA, NCBI-GeneID: 945574); and    -   formate+H⁺=CO₂+H₂ (for example, catalyzed by formate        hydrogenlyase (EC 1.2.1.2 together with EC 1.12.1.2); for E.        coli-fdhF (EC 1.2.1.2), NCBI-GeneID: 948584).

Thus, in a preferred embodiment, the microorganism according to theinvention is characterized in that one or more of the above listedenzyme activities are eliminated or reduced.

One skilled in the art would further recognize that geneticmodifications to regulatory elements in the microorganisms of thepresent invention could lead to improvements in the efficacy by whichthe microorganisms of the present invention convert feedstock toproduct. Within E. coli, such genetic modifications include, but are notlimited to, the group consisting of:

-   -   deleting the fnr gene (NCBI-GeneID: 945908), a global regulator        of anaerobic growth; and    -   deleting the rpoS gene (NCBI-GeneID: 947210), a RNA polymerase,        sigma S (sigma 38) factor.        Thus, in another preferred embodiment the microorganism        according to the invention shows at least one of these        deletions.

A further aspect of the present invention is the use of the recombinantmicroorganism of the present invention for the conversion of glucoseinto acetyl-CoA. Acetyl CoA (also known as acetyl Coenzyme A) inchemical structure is the thioester between coenzyme A (a thiol) andacetic acid and is an important precursor molecule for the production ofuseful metabolites. Acetyl-CoA can then be further converted by therecombinant microorganism into useful metabolites such as L-glutamicacid, L-glutamine, L-proline, L-arginine, L-leucine, succinate andpolyhydroxybutyrate.

Another aspect of the present invention is the use of the recombinantmicroorganism of the present invention that is capable of convertingacetyl-CoA into acetone for the conversion of glucose into acetone.

A further aspect of the present invention is the use of the recombinantmicroorganism of the present invention that is capable of convertingacetyl-CoA into acetone and acetone into isobutene for the conversion ofglucose into isobutene.

Again a further aspect of the present invention is the use of therecombinant microorganism of the present invention that is capable ofconverting acetyl-CoA into acetone and acetone into propene for theconversion of glucose into propene.

Accordingly, the present invention also relates to a method for theproduction of acetone and/or isobutene and/or propene from glucose inwhich the above-mentioned recombinant microorganism is cultivated underconditions allowing for the production of acetone and/or isobuteneand/or propene and in which the acetone and/or isobutene and/or propeneis isolated. The microorganisms are cultivated under suitable cultureconditions allowing the occurrence of the enzymatic reaction(s). Thespecific culture conditions depend on the specific microorganismemployed but are well known to the person skilled in the art. Theculture conditions are generally chosen in such a manner that they allowthe expression of the genes encoding the enzymes for the respectivereactions. Various methods are known to the person skilled in the art inorder to improve and fine-tune the expression of certain genes atcertain stages of the culture such as induction of gene expression bychemical inducers or by a temperature shift.

In another preferred embodiment the method according to the inventionfurthermore comprises the step of collecting gaseous products, inparticular isobutene or propene, degassing out of the reaction, i.e.recovering the products which degas, e.g., out of the culture. Thus in apreferred embodiment, the method is carried out in the presence of asystem for collecting isobutene or propene under gaseous form during thereaction.

As a matter of fact, short alkenes such as isobutene and propene adoptthe gaseous state at room temperature and atmospheric pressure. Themethod according to the invention therefore does not require extractionof the product from the liquid culture medium, a step which is alwaysvery costly when performed at industrial scale. The evacuation andstorage of the gaseous hydrocarbons and their possible subsequentphysical separation and chemical conversion can be performed accordingto any method known to one of skill in the art.

The present invention is further described by reference to the followingnon-limiting FIGURES and examples.

FIG. 1 shows two schemes for the production of acetyl-CoA fromglucose-6-phosphate via the phosphoketolase pathway using either one orboth phosphoketolase activities EC 4.1.2.9 and EC 4.1.2.22.

EXAMPLES General Methods and Materials

Procedure for ligations and transformations are well known in the art.Techniques suitable for use in the following examples may be found inSambrook J., et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor, N.Y., 1989, and Sambrook J., supra.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found in Manual of Methods forGeneral Bacteriology (Philipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhilips, eds).

All reagents and materials used for the growth and maintenance ofbacterial cells were obtained from Sigma-Aldrich Company (St. Louis,Mo.) unless otherwise specified.

TABLE 1 Plasmids used and constructed Plasmid Name Description pKD46Datsenko KA. And Wanner BL., Proceedings of the National Academy ofSciences, 2000, Vol. 97, No. 12, pp. 6640-6645 pCP20 Datsenko KA. AndWanner BL., Proceedings of the National Academy of Sciences, 2000, Vol.97, No. 12, pp. 6640-6645 pGBE0687 Plasmid pGBE0687 presents aresistance gene to apramycin placed under the control of its ownpromoter pGBE0688 Plasmid pGBE0688 presents a resistance gene tospectinomycin placed under the control of its own promoter pGBE0421Plasmid from GeneArt ® (Invitrogen) that encodes for L. lactisphosphoketolase pGBE0123 A modified version of the plasmid pUC18 (NewEngland Biolabs) which contains a modified Multiple Cloning Site (MCS)pGBE0457 Plasmid that allows expression of the L. lactis phosphoketolasepGBE0689 pBluescript II phagemids, Agilent Technologies pGBE0690 ctfAand ctfB genes from Clostridium acetobutylicum cloned into pGBE0689pGBE0691 adc gene from Clostridium acetobutylicum cloned into pGBE0690pGBE0051 pUC19, New England Biolabs pGBE0692 ctfA, ctfB and adc genesfrom Clostridium acetobutylicum cloned into pGBE0051 pGBE0693 thl genefrom from Clostridium acetobutylicum cloned into pGBE0692 pGBE0124 Amodified version of the plasmid pSU18 (Borja Bartolomé, Yolanda Jubete,Eduardo Martinez and Fernando de la Cruz, Gene, 1991, Vol. 102, Issue 1,pp. 75-78) pGBE0096 thl, ctfA, ctfB and adc genes from Clostridiumacetobutylicum cloned into pGBE0124 pGBE0928 Plasmid that allowsexpression of the L. lactis phosphoketolase pGBE1020 Plasmid that allowsexpression of the L. lactis phosphoketolase and acetone productionpGBE1021 Plasmid that allows acetone production

TABLE 2 Strains used and constructed. Strain of Strain origin nameGenotype Construction GBE0129 F-lambda-ilvG-rfb-50 rph-1 Escherichiacoli K12 wild- type MG1655 GBE0170 pKD46 GBE0129 Transformation of thestrain GBE0129 with the pKD46 plasmid GBE0329 pGBE0096 GBE0129Transformation of the strain GBE0129 with the pGBE0096 plasmid GBE0901ΔptsHI::FRT GBE0129 GBE0902 ΔptsHI::FRT pKD46 GBE0901 Transformation ofthe strain GBE0901 with the pKD46 plasmid GBE0903 ΔptsHI::FRTΔzwf_edd_eda::aad⁺ GBE0902 GBE0929 ΔptsHI::FRT GBE0901 Selection of thestrain GBE0901 on MS medium with glucose as the source of carbon.GBE1000 ΔptsHI::FRT Δzwf_edd_eda:: GBE0903. Selection of the strain aad⁺GBE0903 on MS medium with glucose as the source of carbon. GBE1001ΔptsHI::FRT Δzwf_edd_eda:: GBE1000 Transformation of the strain aad⁺pKD46 GBE1000 with the pKD46 plasmid GBE1005_pKD46 ΔptsHI::FRTΔzwf_edd_eda:: GBE1001 aad⁺ ΔpfkA::aac⁺ GBE1005 ΔptsHI::FRTΔzwf_edd_eda:: GBE1005_pKD46. The loss of the pKD46 aad⁺ ΔpfkA::aac⁺plasmid has been verified. GBE1005_p ΔptsHI::FRT Δzwf_edd_eda:: GBE1005Transformation of the strain aad⁺ ΔpfkA::aac⁺ pCP20 GBE1005 with thepCP20 plasmid GBE1006 ΔptsHI::FRT Δzwf_edd_eda::FRT GBE1005_p The lossof the pCP20 ΔpfkA::FRT plasmid has been verified. GBE1010 ΔptsHI::FRTΔzwf_edd_eda::FRT GBE1006 Transformation of the strain ΔpfkA::FRT pKD46GBE1006 with the pKD46 plasmid GBE1014_pKD46 ΔptsHI::FRTΔzwf_edd_eda::FRT GBE1010 ΔpfkA::FRT ΔpfkB::aad⁺ GBE1014 ΔptsHI::FRTΔzwf_edd_eda::FRT GBE1014_pKD46. The loss of the pKD46 ΔpfkA::FRTΔpfkB:: aad⁺ plasmid has been verified. GBE1283 ΔptsHI::FRT GBE0929Successive cultures of the GBE0929 in MS medium with glucose as thesource of carbon. GBE1284 ΔptsHI::FRT pKD46 GBE1283 Transformation ofthe strain GBE1283 with the pKD46 plasmid GBE1287 ΔptsHI::FRTΔzwf_edd_eda::aad⁺ GBE1284 GBE1337 ΔptsHI::FRT Δzwf_edd_eda::aad⁺GBE1287 Transformation of the strain pKD46 GBE1287 with the pKD46plasmid GBE1339 Δzwf_edd_eda::aac⁺ GBE0170 GBE1340 Δzwf_edd_eda::aac⁺pKD46 GBE1339 Transformation of the strain GBE1339 with the pKD46plasmid GBE1341_pKD46 Δzwf_edd_eda::aac⁺ ΔpfkA:: aad⁺ GBE1340 GBE1341Δzwf_edd_eda::aac⁺ ΔpfkA:: aad⁺ GBE1341_pKD46 The loss of the pKD46plasmid has been verified. GBE1341_p Δzwf_edd_eda::aac⁺ ΔpfkA:: aad⁺GBE1341 Transformation of the strain pCP20 GBE1341 with the pCP20plasmid GBE1342 Δzwf_edd_eda::FRT ΔpfkA::FRT GBE1341_p The loss of thepCP20 plasmid has been verified. GBE1343 Δzwf_edd_eda::FRT ΔpfkA::FRTGBE1342 Transformation of the strain pKD46 GBE1342 with the pKD46plasmid GBE1344_pKD46 Δzwf_edd_eda::FRT ΔpfkA::FRT GBE1343 ΔpfkB:: aad⁺GBE1344 Δzwf_edd_eda::FRT ΔpfkA::FRT GBE1344_pKD46 The loss of the pKD46ΔpfkB:: aad⁺ plasmid has been verified. GBE1345 Δzwf_edd_eda::FRTΔpfkA::FRT GBE1344 Transformation of the strain ΔpfkB:: aad⁺ pGBE457GBE1344 with both pGBE96 pGBE0096 and pGB457 plasmids GBE1346 pGBE0096GBE0329 Adaptation of the strain GBE0329 to the MS medium + glucose (2g/L) + Chloramphenicol (25 ug/ml) GBE1347 Δzwf_edd_eda::FRT ΔpfkA::FRTGBE1345 Adaptation of the strain ΔpfkB:: aad⁺ pGBE457 pGBE96 GBE1345 tothe MS medium + glucose (2 g/L) + Chloramphenicol (25 ug/ml) GBE1348ΔptsHI::FRT pGB96 GBE0929 Transformation of the strain GBE0929 with bothpGBE96 and pGB457 plasmids GBE1349 ΔptsHI::FRT Δzwf_edd_eda::FRT GBE1014Transformation of the strain ΔpfkA::FRT ΔpfkB:: aad⁺ GBE1014 with bothpGBE96 pGBE457 pGBE0096 and pGB457 plasmids GBE1350 ΔptsHI::FRT pGB96GBE1348 Adaptation of the strain GBE1348 to the MS medium + glucose (2g/L) + Chloramphenicol (25 ug/ml) GBE1351 ΔptsHI::FRT Δzwf_edd_eda::FRTGBE1349 Adaptation of the strain ΔpfkA::FRT ΔpfkB:: aad⁺ GBE1349 to theMS medium + pGBE457 pGBE0096 glucose (2 g/L) + Chloramphenicol (25ug/ml) GBE1353_pKD46 ΔptsHI::FRT GBE1337 Δzwf_edd_eda::aad+ ΔpfkA:: aac+GBE1353 ΔptsHI::FRT GBE1353_pKD46 The loss of the pKD46Δzwf_edd_eda::aad+ ΔpfkA:: plasmid has been verified. aac+ GBE1353_pΔptsHI::FRT GBE1353 Transformation of the strain Δzwf_edd_eda::aad+ΔpfkA:: GBE1353 with the pCP20 aac+ pCP20 plasmid GBE1368 ΔptsHI::FRTΔzwf_edd_eda:: GBE1353_p The loss of the pCP20 FRT ΔpfkA:: FRT plasmidhas been verified. GBE1371 ΔptsHI::FRT Δzwf_edd_eda:: GBE1368Transformation of the strain FRT ΔpfkA:: FRT pKD46 GBE1368 with thepKD46 plasmid GBE1420_pKD46 ΔptsHI::FRT Δzwf_edd_eda:: GBE1371 FRTΔpfkA:: FRT ΔpfkB:: aad+ GBE1420 ΔptsHI::FRT Δzwf_edd_eda::GBE1420_pKD46 The loss of the pKD46 FRT ΔpfkA:: FRT ΔpfkB:: aad+ plasmidhas been verified. GBE1433 ΔptsHI::FRT Δzwf:: aad+ GBE1284 GBE1436ΔptsHI::FRT Δzwf:: aad+ pKD46 GBE1433 Transformation of the strainGBE1433 with the pKD46 plasmid GBE1441_pKD46 ΔptsHI::FRT Δzwf:: aad+ΔpfkA:: GBE1436 aac+ GBE1441 ΔptsHI::FRT Δzwf:: aad+ ΔpfkA::GBE1441_pKD46 The loss of the pKD46 aac+ plasmid has been verified.GBE1441_p ΔptsHI::FRT Δzwf:: aad+ ΔpfkA:: GBE1441 Transformation of thestrain aac+ pCP20 GBE1441 with the pCP20 plasmid GBE1448 ΔptsHI::FRTΔzwf:: FRT ΔpfkA:: GBE1441_p The loss of the pCP20 FRT plasmid has beenverified. GBE1449 ΔptsHI::FRT Δzwf:: FRT ΔpfkA:: GBE1448 Transformationof the strain FRT pKD46 GBE1448 with the pKD46 plasmid GBE1518_pKD46ΔptsHI::FRT Δzwf:: FRT ΔpfkA:: GBE1449 FRT ΔpfkB:: aad+ GBE1518ΔptsHI::FRT Δzwf:: FRT ΔpfkA:: GBE1518_pKD46 The loss of the pKD46 FRTΔpfkB:: aad+ plasmid has been verified. GBE2252_pKD46 ΔptsHI::FRTΔpfkA:: aad+ GBE1284 GBE2252 ΔptsHI::FRT ΔpfkA:: aad+ GBE2252_pKD46 Theloss of the pKD46 plasmid has been verified. GBE2253 ΔptsHI::FRT ΔpfkA::aad+ pKD46 GBE2252 Transformation of the strain GBE2252 with the pKD46plasmid GBE2256_pKD46 ΔptsHI::FRT ΔpfkA:: aad+ ΔpfkB:: GBE2253 aac+GBE2256 ΔptsHI::FRT ΔpfkA:: aad+ ΔpfkB:: GBE2256_pKD46 The loss of thepKD46 aac+ plasmid has been verified. GBE2262 F-lambda-ilvG-rfb-50 rph-1GBE0129 Transformation of the strain pGB1021 GBE0129 with pGB1021plasmid GBE2263 Δzwf_edd_eda::FRT ΔpfkA::FRT GBE1344 Transformation ofthe strain ΔpfkB:: aad+ pGB1020 GBE1344 with pGB1020 plasmid GBE2264F-lambda-ilvG-rfb-50 rph-1 GBE2262 Adaptation of the strain pGB1021GBE2262 to the MS medium + glucose (2 g/L) + ampicilline (100 ug/ml).GBE2265 Δzwf_edd_eda::FRT ΔpfkA::FRT GBE2263 Adaptation of the strainΔpfkB:: aad+ pGB1020 GBE2263 to the MS medium + glucose (2 g/L) +ampicilline (100 ug/ml). GBE2266 ΔptsHI::FRT pGB1021 GBE1283Transformation of the strain GBE1283 with pGB1021 plasmid GBE2267ΔptsHI::FRT Δzwf_edd_eda:: GBE1420 Transformation of the strain FRTΔpfkA:: FRT ΔpfkB:: aad+ GBE1420 with pGB1020 pGB1020 plasmid GBE2268ΔptsHI::FRT pGB1021 GBE2266 Adaptation of the strain GBE2266 to the MSmedium + glucose (2 g/L) + ampicilline (100 ug/ml). GBE2269 ΔptsHI::FRTΔzwf_edd_eda:: GBE2267 Adaptation of the strain FRT ΔpfkA:: FRT ΔpfkB::aad+ GBE2267 to the MS medium + pGB1020 glucose (2 g/L) + ampicilline(100 ug/ml). GBE2270 ΔptsHI::FRT ΔpfkA:: aad+ ΔpfkB:: GBE2256Transformation of the strain aac+ pGB1020 GBE2256 with pGB1020 plasmidGBE2271 ΔptsHI::FRT Δzwf:: FRT ΔpfkA:: GBE1518 Transformation of thestrain FRT ΔpfkB:: aad+ pGB1020 GBE1518 with pGB1020 plasmid GBE2272ΔptsHI::FRT ΔpfkA:: aad+ ΔpfkB:: GBE2270 Adaptation of the strain aac+pGB1020 GBE2270 to the MS medium + glucose (2 g/L) + ampicilline (100ug/ml). GBE2273 ΔptsHI::FRT Δzwf:: FRT ΔpfkA:: GBE2271 Adaptation of thestrain FRT ΔpfkB:: aad+ pGB1020 GBE2271 to the MS medium + glucose (2g/L) + ampicilline (100 ug/ml). FRT: FLP recognition target

TABLE 3Sequences of bacterial chromosomal regions, genes used, plasmids regions.SEQ Name Nucleotide sequence Description ID NO nucleotideAggctagactttagttccacaacactaaacctataagttggggaaat FRT region SQsequence from acagtgtaggctggagctgcttcgaagttcctatactttctagagaa is 0001strain GBE0901, taggaacttcggaataggaactaaggaggatattcatag underlinedfrom base pairs 2531736 to 2531870 Spectinomycinagagcggccgccaccgcgggaagttcctatactttctagagaatagg FRT SQ resistanceaacttcagctgatagaaacagaagccactggagcacctcaaaaacac regions are 0002cassette catcatacactaaatcagtaagttggcagcatcaccgacgcactttg underlinedcgccgaataaatacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctacatatgagtgaaaaagtgcccgccgagatttcggtgcaactatcacaagcactcaacgtcatcgggcgccacttggagtcgacgttgctggccgtgcatttgtacggctccgcactggatggcggattgaaaccgtacagtgatattgatttgctggtgactgtagctgcaccgctcaatgatgccgtgcggcaagccctgctcgtcgatctcttggaggtttcagcttcccctggccaaaacaaggcactccgcgccttggaagtgaccatcgtcgtgcacagtgacatcgtaccttggcgttatccggccaggcgggaactgcagttcggagagtggcagcgcaaagacatccttgcgggcatcttcgagcccgccacaaccgattctgacttggcgattctgctaacaaaggcaaagcaacatagcgtcgtcttggcaggttcagcagcgaaggatctcttcagctcagtcccagaaagcgatctattcaaggcactggccgatactctgaagctatggaactcgccgccagattgggcgggcgatgagcggaatgtagtgcttactttgtctcgtatctggtacaccgcagcaaccggcaagatcgcgccaaaggatgttgctgccacttgggcaatggcacgcttgccagctcaacatcagcccatcctgttgaatgccaagcgggcttatcttgggcaagaagaagattatttgcccgctcgtgcggatcaggtggcggcgctcattaaattcgtgaagtatgaagcagttaaactgcttggtgccagccaataagaagttcctatactttctagagaataggaacttcgcatgcacgcagcatatgc Apramycinagagcggccgccaccgcgggaagttcctatactttctagagaatagg FRT SQ resistanceaacttcgggttcatgtgcagctccatcagcaaaaggggatgataagt regions are 0003cassette ttatcaccaccgactatttgcaacagtgccgttgatcgtgctatgat underlinedcgactgatgtcatcagcggtggagtgcaatgtcgtgcaatacgaatggcgaaaagccgagctcatcggtcagcttctcaaccttggggttacccccggcggtgtgctgctggtccacagctccttccgtagcgtccggcccctcgaagatgggccacttggactgatcgaggccctgcgtgctgcgctgggtccgggagggacgctcgtcatgccctcgtggtcaggtctggacgacgagccgttcgatcctgccacgtcgcccgttacaccggaccttggagttgtctctgacacattctggcgcctgccaaatgtaaagcgcagcgcccatccatttgcctttgcggcagcggggccacaggcagagcagatcatctctgatccattgcccctgccacctcactcgcctgcaagcccggtcgcccgtgtccatgaactcgatgggcaggtacttctcctcggcgtgggacacgatgccaacacgacgctgcatcttgccgagttgatggcaaaggttccctatggggtgccgagacactgcaccattcttcaggatggcaagttggtacgcgtcgattatctcgagaatgaccactgctgtgagcgctttgccttggcggacaggtggctcaaggagaagagccttcagaaggaaggtccagtcggtcatgcctttgctcggttgatccgctcccgcgacattgtggcgacagccctgggtcaactgggccgagatccgttgatcttcctgcatccgccagaggcgggatgcgaagaatgcgatgccgctcgccagtcgattggctgagctcatgagcggagaacgagatgacgttggaggggcaaggtcgcgctgattgctggggcaacacgtggagcggatcggggattgtctttcttcagctcgctgatgatatgctgacgctcaatgccgaagttcctatactttctagagaataggaacttcgcatgcacgcagcatatg c MCS of theAAGCTTGCGGCCGCGGGGTTAATTAACCTCCTTAGTTTAAACCTAGG The SQ pGB0123CATGCCTCTAGAGGATCCCCGGGTACCGAGCTCGAAttaCCTGCAGG restriction 0004 plasmidAATTC sites for HindIII and EcoRI are underlined. OptimizedTTAATTAATGCATCATCACCACCATCACATGACCGAATATAACAGCG The SQ LactococcusAAGCCTATCTGAAAAAACTGGATAAATGGTGGCGTGCAGCAACCTAT restriction 0005 lactisCTGGGTGCAGGTATGATTTTTCTGAAAGAAAATCCGCTGTTTAGCGT site for phosphoketolaseTACCGGCACCCCGATTAAAGCAGAAAATCTGAAAGCCAATCCGATTG PacI and gene flanked byGTCATTGGGGCACCGTTAGCGGTCAGACCTTTCTGTATGCACATGCA NotI are PacI and NotIAATCGCCTGATTAACAAATATAACCAGAAAATGTTTTATATGGGTGG underlined. restrictionTCCGGGTCATGGTGGTCAGGCAATGGTTGTTCCGAGCTATCTGGATG sitesGTAGCTATACCGAAGCATATCCGGAAATTACCCAGGATCTGGAAGGTATGAGCCGTCTGTTTAAACGTTTTAGCTTTCCGGGTGGTATTGGTAGCCACATGACCGCACAGACACCGGGTAGCCTGCATGAAGGTGGTGAACTGGGTTATGTTCTGAGCCATGCAACCGGTGCAATTCTGGATCAGCCGGAACAAATTGCATTTGCAGTTGTTGGTGATGGTGAAGCAGAAACCGGTCCGCTGATGACCAGCTGGCATAGCATCAAATTTATCAACCCGAAAAACGATGGTGCCATTCTGCCGATTCTGGATCTGAATGGCTTTAAAATCAGCAATCCGACCCTGTTTGCACGTACCAGTGATGTTGATATCCGCAAATTTTTCGAAGGTCTGGGTTATAGTCCGCGTTATATTGAAAACGATGACATCCATGACTACATGGCCTATCATAAACTGGCAGCAGAAGTTTTTGACAAAGCCATTGAAGATATCCATCAGATTCAGAAAGATGCCCGTGAAGATAATCGCTATCAGAATGGTGAAATTCCGGCATGGCCGATTGTTATTGCACGTCTGCCGAAAGGTTGGGGTGGTCCTCGTTATAATGATTGGAGCGGTCCGAAATTTGATGGTAAAGGTATGCCGATCGAACATAGCTTTCGTGCACATCAGGTTCCGCTGCCGCTGAGCAGCAAAAACATGGGCACCCTGCCGGAATTTGTTAAATGGATGACCAGCTATCAGCCGGAAACCCTGTTTAATGCAGATGGTAGCCTGAAAGAAGAACTGCGCGATTTTGCACCGAAAGGTGAAATGCGTATGGCAAGCAATCCGGTTACCAATGGTGGTGTTGATTATAGCAATCTGGTTCTGCCGGATTGGCAAGAATTTGCAAATCCGATTAGCGAAAACAATCGTGGTAAACTGCTGCCGGATACCAATGATAATATGGATATGAACGTGCTGAGCAAATATTTCGCCGAAATTGTTAAACTGAACCCGACCCGTTTTCGTCTGTTTGGTCCGGATGAAACCATGAGCAATCGTTTTTGGGAGATGTTTAAAGTGACCAATCGTCAGTGGATGCAGGTTATCAAAAATCCGAACGATGAGTTTATTAGTCCGGAAGGTCGCATTATTGATAGCCAGCTGAGCGAACATCAGGCAGAAGGTTGGCTGGAAGGTTATACCCTGACCGGTCGTACCGGTGTTTTTGCAAGCTATGAAAGTTTTCTGCGTGTTGTTGATAGCATGCTGACCCAGCACTTTAAATGGATTCGTCAGGCAGCAGATCAGAAATGGCGTCATGATTATCCGAGCCTGAATGTTATTAGCACCAGCACCGTTTTTCAGCAGGATCATAATGGTTATACCCATCAAGATCCGGGTATGCTGACCCATCTGGCAGAGAAAAAAAGCGATTTTATTCGTCAGTATCTGCCTGCAGATGGTAATACCCTGCTGGCCGTTTTTGATCGTGCATTTCAGGATCGCAGCAAAATTAACCATATTGTTGCAAGCAAACAGCCTCGTCAGCAGTGGTTTACCAAAGAAGAAGCAGAAAAACTGGCCACCGATGGTATTGCAACCATTGATTGGGCAAGCACCGCAAAAGATGGTGAAGCCGTTGATCTGGTTTTTGCAAGTGCCGGTGCAGAACCGACCATTGAAACCCTGGCAGCACTGCATCTGGTTAATGAAGTTTTTCCGCAGGCCAAATTTCGCTATGTTAATGTTGTTGAACTGGGTCGTCTGCAGAAAAAGAAAGGTGCACTGAATCAAGAACGCGAACTGAGTGATGAAGAGTTCGAAAAATACTTTGGTCCGAGCGGTACACCGGTTATTTTTGGTTTTCATGGCTATGAAGATCTGATCGAGAGCATCTTTTATCAGCGTGGTCATGATGGTCTGATTGTTCATGGTTATCGTGAAGATGGTGATATTACCACCACCTATGATATGCGTGTTTATAGCGAACTGGATCGTTTTCATCAGGCAATTGATGCAATGCAGGTTCTGTATGTGAATCGTAAAGTTAATCAGGGTCTGGCCAAAGCATTTATTGATCGTATGGAACGTACCCTGGTGAAACATTTTGAAGTTACCCGTAATGAAGGCGTTGATATTCCGGAATTTACCGAATGGGTTTGGAGCGATCTGAAA AAGTAATGAGCGGCCGCMCS of the GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGGCATGCCTAGGTT SQ0006pGB0124 plasmid TAAACTAAGGAGGTTAATTAACCCCGCGGCCGCAAGCTT

TABLE 4 Primer sequence SEQ Name Sequence Description ID NO: 0635Ggttcaattcttcctttagcggc Primer for sequencing the E. coli RC0001chromosomal region including the ptsHI genes. 0638Ccgcaaaaacgacatccggcacg Primer for sequencing the E. coli RC0002chromosomal region including the ptsHI genes. 0633caagtataccctggcttaagtaccgggttagttaa Primer for deletion of the RC0003cttaaggagaatgacAGAGCGGCCGCCACCGCGGG zwf_edd_eda genes. 0634gcaaaaaaacgctacaaaaatgcccgatcctcgat Primer for deletion of the RC0004cgggcattttgacttGCATATGCTGCGTGCATGCG zwf_edd_eda genes 1036Ccgcactttgcgcgcttttccc Primer for sequencing the E. coli RC0005chromosomal region including the zwf_edd_eda genes. 1037Ggtgattttcagtgaggtctcccc Primer for sequencing the E. coli RC0006chromosomal region including the zwf_edd_eda genes. 0629agacttccggcaacagatttcattttgcattccaa Primer for deletion of the pfkARC0007 agttcagaggtagtcAGAGCGGCCGCCACCGCGGG gene 0630gcttctgtcatcggtttcagggtaaaggaatctgc Primer for deletion of the pfkARC0008 ctttttccgaaatcaGCATATGCTGCGTGCATGCG gene. 0619Ggcgctcacgatcttcgcacgcggc Primer for sequencing the E. coli RC0009chromosomal region including the pfkA gene. 0620Ccgcctcatattgctgacaaagtgcgc Primer for sequencing the E. coli RC0010chromosomal region including the pfkA gene. 0631actttccgctgattcggtgccagactgaaatcagc Primer for deletion of the pfkBRC0011 ctataggaggaaatgAGAGCGGCCGCCACCGCGGG gene. 0632gttgccgacaggttggtgatgattcccccaatgct Primer for deletion of the pfkBRC0012 gggggaatgtttttgGCATATGCTGCGTGCATGCG gene. 0621Ccacagcgaccaggcagtggtgtgtcc Primer for sequencing the E. coli RC0013chromosomal region including the pfkB gene. 0622Gcactttgggtaagccccgaaacc Primer for sequencing the E. coli RC0014chromosomal region including the pfkB gene. pUC18_246Ccattcaggctgcgcaactg Primer for sequencing the RC0015phosphoketolase gene from Lactococcus lactis cloned into the pGB0123.pUC18_3800_ Gcgcgttggccgattcattaatgc Primer for sequencing the RC0016rev phosphoketolase gene from Lactococcus lactis cloned into thepGB0123. Pkt CAATCCGACCCTGTTTGCACGTAC Primer for sequencing the RC0017lacto phosphoketolase gene from 700_dirLactococcus lactis cloned into the pGB0123. Pkt_lacto_GCTGCCGGATACCAATGATAATATGG Primer for sequencing the RC0018 mil_dirphosphoketolase gene from Lactococcus lactis cloned into the pGB0123.Pkt_lacto_ CCATATTATCATTGGTATCCGGCAGC Primer for sequencing the RC0019mil_rev phosphoketolase gene from Lactococcus lactis cloned into thepGB0123. 0070 CCCGGGGGATCCAGAATTTAAAAGGAGGGATTPrimer for amplifying the ctfA and RC0020 ctfB genes from Clostridiumacetobutylicum ATCC 824 strain. 0071 CTCGAGGATATCAAGAATTCTTTTTAAACAGCCATPrimer for amplifying the ctfA and RC0021 GGGTCctfB genes from Clostridium acetobutylicum ATCC 824 strain. 1066TGTAAAACGACGGCCAGT General primer for sequencing RC0022 1067CAGGAAACAGCTATGACC General primer for sequencing RC0023 0072CTCGAGGATATCAGGAAGGTGACTTTTATGTTAAA Primer for amplifying the adc geneRC0024 GG from the Clostridium acetobutylicum ATCC 824 strain. 0073GCATGCGTCGACATTAAAAAAATAAGAGTTACC Primer for amplifying the adc geneRC0025 from Clostridium acetobutylicum ATCC 824 strain 1068CCTCACGGCAAAGTCTCAAGC Primer for sequencing the ctfA and RC0026ctfB genes 1069 GCCATGGGTCTAAGTTCATTGGPrimer for sequencing the ctfA and RC0027 ctfB genes 0074CATGATTTTAAGGGGGGTACCATATGCATAAGTTT Primer for amplifying the thl geneRC0028 AA from Clostridium acetobutylicum ATCC 824 strain 0075GTTATTTTTAAGGATCCTTTTTAGCACTTTTCTAG Primer for amplifying the thl geneRC0029 C from Clostridium acetobutylicum ATCC 824 strain 1070GGCAGAAAGGGAGAAACTGTAG Primer for sequencing the acetone RC0030operon from Clostridium acetobutylicum (ATCC 824) 1071TGGAAAGAATACGTGCAGGCGG Primer for sequencing the acetone RC0031operon from Clostridium acetobutylicum (ATCC 824) 1072GATTACGCCAAGCTTGCATGCC Primer for sequencing the acetone RC0032operon from Clostridium acetobutylicum (ATCC 824) 1073CCGGCCTCATCTACAATACTACC Primer for sequencing the acetone RC0033operon from Clostridium acetobutylicum (ATCC 824) 1074CCCATTATTGCTGGGTCAACTCC Primer for sequencing the acetone RC0034operon from Clostridium acetobutylicum (ATCC 824) 1516CCCGGTACCTCATTACTTTTTCAGATCGCTCCAAA Primer for amplifying the RC0035 CCCphosphoketolase gene (YP_003354041.1) from Lactococcus lactis. 1517GGGGAATTCAGGAGGTGTACTAGATGCATCATCAC Primer for amplifying the RC0036CACCATCACATGACC phosphoketolase gene (YP_003354041.1) fromLactococcus lactis. 1994 CCATAGCTCCACCCATACCAGAGAGCPrimer for sequencing the acetone RC0037 operon from Clostridiumacetobutylicum (ATCC 824) 1995 GCTATTATTACGTCAGCATCTCCTGCPrimer for sequencing the acetone RC0038 operon from Clostridiumacetobutylicum (ATCC 824) 1996 GCAGGCGAAGTTAATGGCGTGCPrimer for sequencing the acetone RC0039 operon from Clostridiumacetobutylicum (ATCC 824) 1997 GATACGGGGTAACAGATAAACCATTTCPrimer for sequencing the acetone RC0040 operon from Clostridiumacetobutylicum (ATCC 824) 1998 CCCTTTCTGCCTTTAATTACTACAGGPrimer for sequencing the acetone RC0041 operon from Clostridiumacetobutylicum (ATCC 824) 1999 GCATCAGGATTAAATGACTGTGCAGCPrimer for sequencing the acetone RC0042 operon from Clostridiumacetobutylicum (ATCC 824) 2000 GGACTAGCGCCCATTCCAACTATTCCPrimer for sequencing the acetone RC0043 operon from Clostridiumacetobutylicum (ATCC 824) 2001 GCTGCAAGGCGATTAAGTTGGGTAACGCCPrimer for sequencing the acetone RC0044 operon from Clostridiumacetobutylicum (ATCC 824) 2002 GCATTGCGTGTACAAGAGTAACGAGPrimer for sequencing the acetone RC0045 operon from Clostridiumacetobutylicum (ATCC 824) 2003 CCTGTCCAAGCTTCATGTACGGPrimer for sequencing the acetone RC0046 operon from Clostridiumacetobutylicum (ATCC 824) 1109 GCGCAAGATCATGTTACCGGTAAAATAACCATAAAPrimer for deletion of the zwf gene. RC0047GGATAAGCGCAGATAGCATATGCTGCGTGCATGCG 1110 CGCCTGTAACCGGAGCTCATAGGGPrimer for sequencing the E. coli RC0048chromosomal region including the zwf gene.

Chromosomal Integration for Gene Knockouts.

To integrate DNA into a specific region of the chromosome, homology ofthe inserting DNA to the targeted chromosomal site and a selectablemarker are required. It is advantageous if the marker can be easilyremoved after integration. The FRT/Flp recombinase system provides amechanism to remove the marker. The FRT sites are recognition sites forthe Flp recombinases. Flp is a site specific recombinase, which excisesthe intervening DNA from the directly repeated recognition sites.

The integration cassette containing homologous arms to the targetedchromosomal site and encoding a selectable marker flanked by FRT(Datsenko K A. And Wanner B L., Proceedings of the National Academy ofSciences, 2000, Vol. 97, No. 12, pp. 6640-6645) sites is transformedinto target cells harboring pKD46 (Datsenko K A. And Wanner B L.,Proceedings of the National Academy of Sciences, 2000, Vol. 97, No. 12,pp. 6640-6645). Successful integrants are selected by growth of thecells in the presence of the antibiotic. Subsequently, pKD46 is curedfrom the cells and the recombinase plasmid is then introduced into theintegrants for removal of the antiobiotic gene. Strains containing a FRTcassette are transformed with the pCP20 plasmid that encodes Flprecombinase (Datsenko K A. And Wanner B L., Proceedings of the NationalAcademy of Sciences, 2000, Vol. 97, No. 12, pp. 6640-6645). Afterremoval of the integrated marker, the recombinase plasmids are curedfrom the strain.

Example 1: Construction of Strain GBE1014

The purpose of this section is to describe the construction of anEscherichia coli strain, named GBE1014, for which the PEP-dependentglucose uptake is inactivated by deletion of the PTS transport genes,the ATP-dependent glucose uptake is enabled, the Embden-Meyerhof-Parnaspathway (EMPP) is inactivated by deletion of the phosphofructokinasegenes, and the pentose phosphate pathway (PPP) is inactivated bydeletion of the glucose-6-phosphate dehydrogenase gene.

Construction started with strain GBE0901. GBE0901 is an Escherichiacoli, (Migula) Castellani and Chalmers, strain MG1655 (ATCC #700926)where the original nucleotidic sequence, from by 2531736 to 2533865(NCBI genome database), included the ptsH and ptsl genes, was replacedby SEQ SQ0001. This deletion affects the PEP-dependentphosphotransferase system (PTS), resulting in the PEP-dependent glucoseuptake to be inactivated in strain GBE0901. Deletion of the ptsHI geneswas verified by PCR and oligonucleotides 0635 and 0638 (given as SEQRC0001 and RC0002, respectively) were used as primers. The resulting 0.4Kbp PCR product was sequenced using the same primers.

Strain GBE0901 was cultivated in LB medium and GBE0901 cells were madeelectrocompetent. Electrocompetent GBE0901 cells were transformed with aplasmid named pKD46 (Datsenko K A. And Wanner B L., Proceedings of theNational Academy of Sciences, 2000, Vol. 97, No. 12, pp. 6640-6645) andthen plated on LB plates containing ampicilline (100 ug/ml). Plates wereincubated overnight at 30° C. Transformation of GBE0901 cells withplasmid pKD46 generated strain GBE0902. The plasmid pGBE0688 presents aresistance gene to spectinomycin placed under the control of its ownpromoter. The sequence of this resistance cassette is indicated in table3 (SEQ SQ0002).

Plasmid pGBE0688 was used as a template with primers 0633 and 0634(given as SEQ RC0003 and RC0004, respectively) to generate a 1.3 Kbp PCRproduct. This 1.3 Kbp PCR product was transformed into electrocompetentGBE0902 bacteria and the transformation mixture was then plated on LBplates containing spectinomycin (50 ug/ml) and incubated overnight at37° C. to generate strain GBE0903. In strain GBE0903 the DNA sequencecomposed by the zwf, edd, and eda genes were deleted. These genesrespectively code for a glucose-6-phosphate dehydrogenase, a6-phosphogluconate dehydratase, and a 2-keto-3-deoxy-6-phosphogluconatealdolase. This deleted DNA sequence including the zwf, edd, and edagenes was replaced by a spectinomycin resistance cassette. In order tocheck the effective deletion of the zwf, edd, and eda genes, a PCRamplification was performed with primers 1036 and 1037 (given as SEQRC0005 and RC0006, respectively). A final 1.9 Kbp PCR product wasobtained. This 1.9 Kbp PCR product was sequenced with the same primers1036 and 1037.

Strain GBE0903 was then plated on LB plates, incubated at 37° C. andisolated colonies were screened on MS plates (Richaud C.,Mengin-Leucreulx D., Pochet S., Johnson E J., Cohen G N. and MarHere P;The Journal of Biological Chemistry; 1993; Vol. 268; No. 36; pp.26827-26835) with glucose as the source of carbon (2 g/L). After 48hours of incubation at 37° C., colonies became visible and weretransferred to an MS liquid medium supplied with glucose (2 g/L). Thisovernight incubation at 37° C. induced the loss of the pKD46 plasmid.One isolate had a doubling time of 7 hours and was named GBE1000.

Strain GBE1000 was made electrocompetent. GBE1000 electrocompetent cellswere transformed with plasmid pKD46, and then plated on LB platessupplied with ampicilline (100 ug/ml). Plates were incubated overnightat 30° C. Transformation of GBE1000 cells with plasmid pKD46 generatedstrain GBE1001.

The plasmid pGBE0687 presents a resistance gene to apramycin placedunder the control of its own promoter. The sequence of this resistancecassette is indicated in table 3 (SEQ SQ0003).

The plasmid pGBE0687 was used as a template along with primers 0629 and0630 (given as SEQ RC0007 and RC0008, respectively) to generate a 1.2Kbp PCR product. The resulting 1.2 Kbp PCR product was transformed intoelectrocompetent GBE1001 bacteria and the transformation mixture wasplated on LB plates containing apramycin (50 ug/ml). Plates were thenincubated overnight at 37° C. to generate a new strain namedGBE1005_pKD46. In Strain GB1005_pKD46 the phosphofructokinase gene pfkA,was deleted and was replaced by the apramycin resistance cassette. Tocheck that the deletion of the pfkA gene occurred, a PCR amplificationwas performed with primers 0619 and 0620 (given as SEQ RC0009 andRC0010, respectively). This 1.7 Kbp PCR product was sequenced with thesame primers 0619 and 0620. In order to check the loss of the plasmidpKD46, the strain GBE1005_pKD46 was plated on LB plates and incubatedovernight at 42° C. The loss of the plasmid pKD46 was verified byplating isolated colonies on LB plates containing ampicilline (100ug/ml), incubated overnight at 30° C., and on LB plates incubatedovernight at 37° C. The resulting strain grew on LB plates incubated at37° C. and was named GBE1005. GBE1005 cells did not grow on LB platessupplied with ampicilline (100 ug/ml).

The spectinomycin cassette was located at the corresponding loci of thezwf_edd_eda genes and the apramycin cassette was located at thecorresponding loci of the pfkA genes. In order to excise the resistantcassettes containing the spectinomycin and apramycin resistance genes,the strain GBE1005 was transformed with the plasmid pCP20 (Datsenko K A.And Wanner B L., Proceedings of the National Academy of Sciences, 2000,Vol. 97, No. 12, pp. 6640-6645) to obtain the strain GBE1005_p. Afterovernight incubation on LB plates containing ampicilline (100 ug/ml) at30° C., isolated colonies were restreaked on LB plates supplied withampicilline (100 ug/ml) and incubated overnight at 30° C. Isolatedcolonies were then plated on LB plates and incubated overnight at 42° C.which caused the loss of the pCP20 plasmid. Then, in order to check theeffective excision of the two resistant cassettes and the loss of thepCP20 plasmid, isolated colonies were streaked out on LB platescontaining spectinomycin (50 ug/ml), incubated overnight at 37° C., onLB plates containing apramycin (50 ug/ml), incubated overnight at 37°C., on LB plates containing ampicilline (100 ug/ml), incubated overnightat 30° C. and on LB plates, incubated overnight at 37° C. The resultingstrain grew on LB plates incubated at 37° C. and was named GBE1006.GBE1006 cells did not grow on LB plates containing spectinomycin (50ug/ml), on LB plates containing apramycin (50 ug/ml), and on LB platessupplied with ampicilline (100 ug/ml).

Strain GBE1006 was made electrocompetent, and GBE1006 electrocompetentcells were transformed with plasmid pKD46. Transformant cells were thenplated on LB plates containing ampicilline (100 ug/ml) and plates wereincubated overnight at 30° C. to obtain a new strain named GBE1010. APCR product was generated by using the plasmid pGBE0688 as a templateand the oligonucleotides 0631 and 0632 (given as SEQ RC0011 and RC0012,respectively) as primers. The resulting 1.3 Kbp PCR product wastransformed into electrocompetent GBE1010 bacteria and thetransformation mixture was plated on LB plates containing spectinomycin(50 ug/ml). Plates were incubated overnight at 37° C. to generate strainGBE1014_(—) pKD46. In Strain GBE1014_(—) pKD46 the phosphofructokinasegene pfkB, was deleted and the deleted DNA sequence was replaced by acassette containing the spectinomycin resistance gene. To check that thedeletion of the pfkB gene occurred, a PCR amplification was performedwith primers 0621 and 0622 (given as SEQ RC0013 and RC0014,respectively). This final 2.2 Kbp PCR product was sequenced by using thesame primers 0621 and 0622.

In order to induce the loss of the plasmid pKD46, strain GBE1014_(—)pKD46 was plated on LB plates and plates were incubated overnight at 42°C. The loss of the plasmid pKD46 was checked by plating isolatedcolonies on LB plates supplied with ampicilline (100 ug/ml), incubatedovernight at 30° C., and on LB plates incubated overnight at 37° C. Theresulting strain growing on LB plates incubated at 37° C. was namedGBE1014. GBE1014 cells did not grow on LB plates supplied withampicilline (100 ug/ml).

Example 2: Construction of Strain GBE0929

Strain GBE0901 was plated on LB plates at 37° C. and isolated colonieswere screened on MS plates with glucose as the source of carbon (2 g/L).After 48 hours of incubation at 37° C., colonies became visible. Oneisolate had a doubling time of 5 hours and was named GBE0929.

Example 3: Construction of Strain GBE1344

Construction started with the strain named GBE0129. GBE0129 is an MG1655Escherichia coli bacteria (ATCC #700926).

Strain GBE0129 was cultivated in LB medium and GBE0129 cells were madeelectrocompetent. Electrocompetent GBE0129 cells were transformed withplasmid pKD46, and then transformants were plated on LB platescontaining ampicilline (100 ug/ml). Plates were incubated overnight at30° C. to generate a new strain named GBE0170.

A PCR product was generated using plasmid pGBE0687 as a template andoligonucleotides 0633 and 0634 (given as SEQ RC0003 and RC0004,respectively) as primers. The resulting 1.2 Kbp PCR product wastransformed into electrocompetent GBE0170 bacteria and thetransformation mixture was plated on LB plates containing apramycin (50ug/ml). Plates were incubated overnight at 37° C. This incubationtriggered the loss of the pKD46 plasmid and led to the creation of a newstrain named GBE1339. In Strain GBE1339 the glucose-6-phosphatedehydrogenase encoded by the zwf gene, the 6-phosphogluconatedehydratase encoded by the edd gene and the2-keto-3-deoxy-6-phosphogluconate aldolase encoded by the eda gene wereinactive. The sequential zwf, edd, and eda genes were deleted andreplaced by a cassette containing the apramycin resistance gene. Tocheck that the deletion of the zwf, edd, and eda genes was effective, aPCR amplification was performed with primers 1036 and 1037 (given as SEQRC0005 and RC0006, respectively). This 1.8 Kbp PCR product was sequencedwith the same primers 1036 and 1037.

Strain GBE1339 was made electrocompetent, and GBE1339 electrocompetentcells were transformed with plasmid pKD46. Transformants were thenplated on LB plates supplied with ampicilline (100 ug/ml). Plates wereincubated overnight at 30° C. to generate strain GBE1340. A PCR productwas performed by using plasmid pGBE0688 as a template andoligonucleotides 0629 and 0630 (given as SEQ RC0007 and RC0008,respectively) as primers. The resulting 1.3 Kbp PCR product wastransformed into electrocompetent GBE1340 bacteria and thetransformation mixture was plated on LB plates containing spectinomycin(50 ug/ml). Plates were incubated overnight at 37° C. to generate strainGBE1341_pKD46. In Strain GB1341_pKD46 the pfka gene coding for aphosphofructokinase was replaced by the spectinomycin resistance gene.To check that the deletion of the pfkA gene was effective, a PCRamplification was performed with primers 0619 and 0620 (given as SEQRC0009 and RC0010, respectively). This 1.8 Kbp PCR product was sequencedwith the same primers 0619 and 0620. In order to induce the loss of theplasmid pKD46 for the strain GBE1341_pKD46, GBE1341_pKD46 cells wereplated on LB plates and incubated at 42° C. The loss of the plasmidpKD46 was verified by plating isolated colonies on LB plates suppliedwith ampicilline (100 ug/ml), incubated overnight at 30° C. and on LBplates incubated overnight at 37° C. The resulting strain growing on LBplates incubated at 37° C. was GBE1341. GBE1341 did not grow on LBplates supplied with ampicilline (100 ug/ml).

In order to excise the resistant cassettes containing the apramycin andspectinomycin resistance genes, which were respectively located in theformer loci of the zwf_edd_eda and pfkA genes, the strain GBE1341 wastransformed with plasmid pCP20 to obtain a new strain named GBE1341_p.After overnight incubation on LB plates containing ampicilline (100ug/ml) at 30° C., isolated colonies were restreaked on LB platessupplied with ampicilline (100 ug/ml) for another overnight incubationat 30° C. Isolated colonies were then plated on LB plates and incubatedovernight at 42° C. This incubation at 42° C. triggered the loss of thepCP20 plasmid. Eventually in order to check excision of the tworesistant cassettes and the loss of the pCP20 plasmid, isolated colonieswere streaked out on LB plates containing spectinomycin (50 ug/ml) andincubated overnight at 37° C., on LB plates supplied with apramycin (50ug/ml) and incubated overnight at 37° C., on LB plates containingampicilline (100 ug/ml) and incubated overnight at 30° C. and on LBplates incubated overnight at 37° C. The generated strain growing on LBplates incubated at 37° C. was named GBE1342. GBE1342 cells did not growon LB plates supplied with spectinomycin (50 ug/ml), on LB platessupplied with apramycin (50 ug/ml) and on LB plates containingampicilline (100 ug/ml).

Strain GBE1342 was made electrocompetent, and GBE1342 cells weretransformed with plasmid pKD46. Transformants were then plated on LBplates supplied with ampicilline (100 ug/ml) and incubated overnight at30° C. to obtain strain GBE1343. A PCR product was performed and usedthe plasmid pGBE0688 as a template and oligonucleotides 0631 and 0632(given as SEQ RC0011 and RC0012, respectively) as primers. The resulting1.3 Kbp PCR product was transformed into electrocompetent GBE1343bacteria and the transformation mixture was plated on LB platescontaining spectinomycin (50 ug/ml) followed by an overnight incubationat 37° C. A new strain was generated and named GBE1344_(—) pKD46. InStrain GBE1344_(—) pKD46 the pfkb gene coding for a phosphofructokinasewas deleted and replaced by the spectinomycin resistance cassette. Tocheck that the deletion of the pfkB gene was effective, a PCRamplification was performed with primers 0621 and 0622 (given as SEQRC0013 and RC0014, respectively). The 2.2 Kbp PCR product obtained wassequenced with the same primers 0621 and 0622.

In order to induce the loss of the plasmid pKD46, the strain GBE1344_(—)pKD46 was plated on LB plates and incubated overnight at 42° C. The lossof the plasmid pKD46 was checked by plating isolated colonies on LBplates containing ampicilline (100 ug/ml) and incubated overnight at 30°C. and on LB plates incubated overnight at 37° C. The generated straingrowing on LB plates incubated at 37° C. was named GBE1344. GBE1344cells did not grow on LB plates containing ampicilline (100 ug/ml).

Example 4: Construction of Plasmid pGBE0457

The purpose of this section is to describe the construction of a plasmidthat allows the expression of phosphoketolase YP_003354041.1 fromLactococcus lactis in E. coli strains.

The plasmid pGBE0123 is a modified version of the plasmid pUC18 (NewEngland Biolabs) and contains a modified Multiple Cloning Site (MCS).The original MCS from pUC18 (from HindIII restriction site to EcoRIrestriction site) was replaced by the sequence SQ0004 (table 3). Theplasmid pGB0123 allows expression of two recombinant proteins under thecontrol of the Plac promoter.

Plasmid from Phosphoketolase Phosphoketolase Organism of GeneArt ® geneID protein ID origin (Invitrogen) 8679043 YP_003354041.1 LactococcuspGBE0421 lactis subsp. lactis KF147

The L. lactis phosphoketolase gene was codon-optimized by GeneArt®(Invitrogen) for optimal expression in Escherichia coli. In addition, aHis-tag was added at the 5′ position of the gene and an additional stopcodon was added at the 3′ position (SQ0005). The gene construction isflanked by PacI and NotI restriction sites and provided within plasmidpGBE0421.

For cloning experiment, PCR products and restriction fragments were gelpurified using QIAquick gel Extraction kit (Qiagen). Restriction enzymesand T4 DNA ligase (New England Biolabs, Beverly, Mass.) were usedaccording to manufacturer's recommendations.

Plasmid pGBE0421 was digested with the restriction enzymes PacI and NotIto create a 2.6 Kbp product. The pGB0123 plasmid was digested as wellwith restriction enzymes, PacI and NotI and ligated to the 2.6 Kbprestriction fragment. The resulting plasmid (pGBE0457) was sequencedwith primers 1061, 1062, 1063, 1064 and 1065 (given as SEQ RC0015,RC0016, RC0017, RC0018 and RC0019 respectively).

The expression of the phosphoketolase from Lactococcus lactis waschecked on a protein gel, after purification of the recombinant proteinusing a His trap (Protino Ni-IDA 1000 kit, Macherey Nagel). Purificationwas processed according to the manufacturer's recommendations. Enzymaticassay, with purified enzyme, was also performed in order to detectphosphoketolase activity on two different substrates:xylulose-5-phosphate and fructose-6-phosphate. The experimentalprocedure was the same than the one used by Leo Meile et al., (Journalof Bacteriology, May 2001, p. 2929-2936), except that the pH of thesolution was 7.5 and 1 mM of MgCl2 was added. For this enzymatic assay,10 μg of purified protein was added to the 75 μl of the reaction. Thespecific activity (μmol of Acetyl-P formed/min/mg protein) was 2815μmol/min/mg protein and 1941 μmol/min/mg protein forD-xylulose-5-phosphate and D-fructose-6-phosphate, respectively.

Example 5: Construction of Plasmid pGBE0096

The construction of the plasmids responsible for acetone production inE. coli was based on the plasmid construction described in Bermejo L L.,Welker N E. and Papoutsakis E T., Applied and EnvironmentalMicrobiology, 1998, Vol. 64, No. 3, pp. 1076-1085.

The strain Clostridium acetobutylicum was ordered (ATCC 824). Thegenomic DNA from this strain is made up of a bacterial chromosome and aplasmid named pSOL1. The ctfA and ctfB genes were PCR amplified from thepSOL1 plasmid with primers 0070 and 0071 (given as SEQ RC0020 andRC0021, respectively). A BamHI restriction site at the 5′ end of the PCRproduct and an EcoRV restriction site at the 3′ end of the PCR productwere introduced. The resulting 1.3 Kbp PCR product was digested with therestriction enzymes BamHI and EcoRV, then ligated with the pGB0689(pBluescript II phagemids, Agilent Technologies) which was digested aswell with restriction enzymes, BamHI and EcoRV. The resulting plasmid(pGBE0690) was sequenced with primers 1066 and 1067 (given as SEQ RC0022and RC0023, respectively).

The adc gene and the gene terminator were PCR amplified from the pSOL1plasmid with primers 0072 and 0073 (given as SEQ RC0024 and RC0025,respectively). PCR amplification allowed inserting an EcoRV restrictionsite at the 5′ end and a SalI restriction site at the 3′ end. Theresulting 0.8 Kbp PCR product was digested with the restriction enzymesEcoRV and SalI. The pGBE0690 plasmid was digested as well withrestriction enzymes, EcoRV and SalI and then ligated with the 0.8 KbpPCR product. The resulting plasmid (pGBE0691) was sequenced with primers1066, 1067, 1068 and 1069 (given as SEQ RC0022, RC0023, RC0026 andRC0027, respectively).

Plasmid pGBE0691 was digested with the restriction enzymes BamHI andSalI to create a 2.2 Kbp product. The 2.2 Kbp restriction fragmentcontained the ctfA, ctfB and adc genes. The pGBE0051 plasmid (pUC19, NewEngland Biolabs) was digested as well with restriction enzymes, BamHIand SalI, and was then ligated with the 2.2 Kbp restriction fragment.The resulting plasmid (pGBE0692) was sequenced with primers 1066, 1067,1068 and 1069 (given as SEQ RC0022, RC0023, RC0026 and RC0027,respectively).

The thl gene and its corresponding thl promoter from Clostridiumacetobutylicum (ATCC 824) genomic DNA were PCR amplified with primers0074 and 0075 (given as SEQ RC0028 and RC0029, respectively). PCRamplification allowed inserting a KpnI restriction site at the 5′ endand a BamHI restriction site at the 3′ end. The resulting 1.4 Kbp PCRproduct was digested with the restriction enzymes KpnI and BamHI, andlikewise for the plasmid pGBE0692. The digested pGBE0692 plasmid wasligated with the 1.4 Kbp PCR product. The resulting plasmid, pGBE0693,was sequenced with primers 1066, 1067, 1068, 1069, 1070, 1071, 1072,1073 and 1074 (given as SEQ RC0022, RC0023, RC0026 RC0027, RC0030,RC0031, RC0032, RC0033 and RC0034, respectively).

The plasmid pGBE0124 is a modified version of the plasmid pSU18 (BorjaBartolomé, Yolanda Jubete, Eduardo Martinez and Fernando de la Cruz,Gene, 1991, Vol. 102, Issue 1, pp. 75-78) and it contains a modifiedMultiple Cloning Site (MCS). The original MCS from pSU18 (from EcoRIrestriction site to HindIII restriction site) was replaced by thesequence SEQ SQ0006 (table 3). The plasmid pGB0124 allows expression oftwo recombinant proteins under the control of the Plac promoter. PlasmidpGBE0693 was digested with the restriction enzymes KpnI and SalI tocreate a 3.5 Kbp product. The pGBE0124 plasmid was digested as well withrestriction enzymes KpnI and SalI, and then ligated to the 3.5 Kbprestriction fragment. The resulting plasmid (pGBE0096) was sequencedwith primers 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073 and 1074(given as SEQ RC0022, RC0023, RC0026 RC0027, RC0030, RC0031, RC0032,RC0033 and RC0034, respectively).

Example 6: Acetone Production by the Strains GBE1346 and GBE1347

Description of Plasmid Transformation into Relevant Strains

The strain GBE0129 was made electrocompetent, and GBE0129electrocompetent cells were transformed with plasmid pGBE0096.Transformants were then plated on LB plates containing chloramphenicol(25 ug/ml) and plates were incubated overnight at 30° C. to generatestrain GBE0329.

Strain GBE1344 was made electrocompetent, and GBE1344 electrocompetentcells were transformed with both plasmids pGBE0457 and pGBE0096.Transformants were then plated on LB plates supplied with ampicilline(100 ug/ml) and chloramphenicol (25 ug/ml). Plates were incubatedovernight at 30° C. to obtain strain GBE1345.

Isolated colonies from strains GBE0329 and GBE1345 were screened on MSplates containing glucose as the source of carbon (2 g/L) andchloramphenicol (25 ug/ml). These plates were incubated at 30° C. toobtain strains GBE1346 and GBE1347 respectively. After 4 days ofincubation at 30° C., colonies were transferred to MS liquid mediumcontaining glucose (2 g/L) and chloramphenicol (25 ug/ml) and incubated3 days at 30° C.

Description of Flasks Conditions

For the fermentation experiments, a MS medium with 200 mM of dipotassiumphosphate was used instead of 50 mM dipotassium phosphate. The resultedmedium was named MSP.

400 ml of MSP liquid medium containing glucose (10 g/L) andchloramphenicol (25 ug/ml), were inoculated either with pre culture ofstrain GBE1346 or with pre culture of strain GBE1347. The initial OD₆₀₀was 0.1. The 400 ml of culture were incubated in 500 ml bottles, sealedwith a screw cap, at 30° C., 170 rpm of speed. 2 ml aliquots were takenafter 1 day, 2 days, 3 days, 6 days, 7 days and 8 days. For each aliquotsamples, bottles were open during 10 seconds.

Description of Analytical Methods

Aliquots were filtered and the glucose concentration was determined withthe glucose (HK) Assay kit (GAHK20-1KT, Sigma) according tomanufacturer's recommendations. The acetone concentration was determinedby gas chromatography using Gas chromatograph 450-GC (Bruker) and thefollowing program:

-   -   Column: DB-WAX (123-7033, Agilent Technologies)    -   Injector Split/Splitless: T°=250° C.    -   Oven:        -   80° C. for 6 minutes        -   10° C. per minutes until 220° C.        -   220° C. for 7 minutes        -   Column flow: 1.5 ml/minute (Nitrogen)    -   Detector FID: T°=300° C.

Results

The ratio [acetone] produced (mM)/[glucose] consumed (mM) is higher forthe strain GBE1347 than for the strain GBE1346.

Example 7: Acetone Production by the Strains GBE1350 and GBE1351

Description of Plasmid Transformation into Relevant Strains

Strain GBE0929 was made electrocompetent, and GBE0929 electrocompetentcells were transformed with a plasmid named pGBE0096. Transformants werethen plated on LB plates supplied with chloramphenicol (25 ug/ml) andplates were incubated overnight at 30° C. to obtain strain GBE1348.

Strain GBE1014 were made electrocompetent, transformed with bothplasmids pGBE0457 and pGBE0096. Transformants were then plated on LBplates supplied with ampicilline (100 ug/ml) and chloramphenicol (25ug/ml) and plates were incubated overnight at 30° C. to obtain strainGBE1349.

Isolated colonies from strains GBE1348 and GBE1349 were screened on MSplates containing glucose as the source of carbon (2 g/L) andchloramphenicol (25 ug/ml). These plates were incubated 4 days at 30° C.to obtain strain GBE1350 and GBE1351 respectively. Isolated colonieswere then transferred to MS liquid medium containing glucose (2 g/L) andchloramphenicol (25 ug/ml). GBE1350 and GBE1351 Cells were incubated at30° C.

Description of Flasks Conditions

400 ml of MSP medium containing glucose (10 g/L) and chloramphenicol (25ug/ml) were inoculated either with pre-culture of strain GBE1350 or withpre-culture of strain GBE1351. The initial OD₆₀₀ was 0.1. The 400 ml ofculture were incubated in 500 ml bottles, sealed with a screw cap, at30° C., 170 rpm of speed. 2 ml aliquots were taken after 1 day, 2 days,3 days, 6 days, 7 days and 8 days. For each aliquot samples, bottleswere opened during 10 seconds.

Description of Analytical Methods

Aliquots were filtered and the glucose concentration was determined withthe glucose (HK) Assay kit (GAHK20-1KT, Sigma) according tomanufacturer's recommendations. The acetone concentration was determinedby gas chromatography using Gas chromatograph 450-GC (Bruker) and thefollowing program:

-   -   Column: DB-WAX (123-7033, Agilent Technologies)    -   Injector Split/Splitless: T°=250° C.    -   Oven:        -   80° C. for 6 minutes        -   10° C. per minutes until 220° C.        -   220° C. for 7 minutes        -   Column flow: 1.5 ml/minute (Nitrogen)    -   Detector FID: T°=300° C.

Results

The ratio [acetone] produced (mM)/[glucose] consumed (mM) was higher forthe strain GBE1351 than for the strain GBE1350.

TABLE OF RESULTS I GBE1346 GBE1347 GBE1350 GBE1351 PEP dependentglucose + + − − uptake (ptsHI) Heterologous − + − + phosphoketolase(pkt) EMPP (pfkAB) + − + − PPP (zwf edd eda) + − + − Heterologousfructose − − − − bisphosphatase (fbp) Heterologous acetone + + + +pathway (thl ctfAB adc) [acetone]_(produced)/ 0.02 >0.02 0.04 0.14[glucose]_(consumed)

Example 8: Construction of the Plasmid pGBE1020

The purpose of this section is to describe the construction of a plasmidthat allows the expression of phosphoketolase YP_003354041.1 fromLactococcus lactis and also allows the production of acetone in E. colistrains.

The L. Lactis phosphoketolase gene was PCR amplified from the pGBE0421plasmid with primers 1516 and 1517 (given as SEQ RC0035 and RC0036,respectively).

PCR amplification allowed inserting an EcoRI restriction site and aRibosome Binding Site (RBS) at the 5′ end and a KpnI restriction site atthe 3′ end. The resulting 2.5 Kbp PCR product was digested with therestriction enzymes EcoRI and KpnI. The pGBE0123 plasmid was digested aswell with restriction enzymes, EcoRI and KpnI and then ligated with the2.5 Kbp PCR product. The resulting plasmid (pGBE0928) was sequenced withprimers 1061, 1062, 1063, 1064 and 1065 (given as SEQ RC0015, RC0016,RC0017, RC0018 and RC0019, respectively).

Plasmid pGBE0096 was digested with the restriction enzymes KpnI and NotIto create a 3.6 Kbp product. The pGBE0928 plasmid was digested as wellwith restriction enzymes, KpnI and NotI and ligated to the 3.6 Kbprestriction fragment. The resulting plasmid (pGBE1020) was sequencedwith primers 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002 and2003 (given as SEQ RC0037, RC0038, RC0039, RC0040, RC0041, RC0042,RC0043, RC0044, RC0045 and RC0046, respectively).

Example 9: Construction of the Plasmid pGBE1021

The purpose of this section is to describe the construction of a plasmidthat allows the production of acetone in E. coli strains.

Plasmid pGBE0096 was digested with the restriction enzymes KpnI and NotIto create a 3.6 Kbp product.

The pGBE0123 plasmid was digested as well with restriction enzymes, KpnIand NotI and then ligated with the 3.6 Kbp PCR product. The resultingplasmid (pGBE1021) was sequenced with primers 1994, 1995, 1996, 1997,1998, 1999, 2000, 2001, 2002 and 2003 (given as SEQ RC0037, RC0038,RC0039, RC0040, RC0041, RC0042, RC0043, RC0044, RC0045 and RC0046,respectively).

Example 10: Acetone Production by the Strains GBE2264 and GBE2265

Description of Plasmid Transformation into Relevant Strains

The strain GBE0129 was made electrocompetent, and GBE0129electrocompetent cells were transformed with plasmid pGB1021.Transformants were then plated on LB plates containing ampicilline (100ug/ml) and plates were incubated overnight at 30° C. to generate strainGBE2262.

Strain GBE1344 was made electrocompetent, and GBE1344 electrocompetentcells were transformed with the plasmid pGBE1020. Transformants werethen plated on LB plates supplied with ampicilline (100 ug/ml). Plateswere incubated overnight at 30° C. to obtain strain GBE2263.

Isolated colonies from strains GBE2262 and GBE2263 were screened on MSplates containing glucose as the source of carbon (2 g/L) andampicilline (100 ug/ml). These plates were incubated at 30° C. to obtainstrains GBE2264 and GBE2265, respectively. After 4 days of incubation at30° C., colonies were transferred to MS liquid medium containing glucose(2 g/L), yeast extract (0.1 g/L) and ampicilline (100 ug/ml) andincubated 3 days at 30° C.

Description of Flasks Conditions

MSP liquid medium (200 ml) containing glucose (10 g/L), yeast extract(0.1 g/L) and ampicilline (100 ug/ml), were inoculated either with preculture of strain GBE2264 or with pre culture of strain GBE2265. Theinitial OD₆₀₀ was 0.1. The 200 ml of culture was incubated in 250 mlbottles, sealed with a screw cap, at 30° C., 170 rpm of speed. Aliquots(2 ml) were taken after 1 day, 2 days, 4 days, 5 days and 6 days. Foreach aliquot sample, the bottle was open for 10 seconds.

Description of Analytical Methods

Aliquots were filtered and the glucose concentration was determined byHPLC analysis using the Agilent HPLC (1260 Infinity) and a Hi-PlexColomn (Agilent, PL1170-6830) with a guard column (Agilent, PL Hi-Plex HGuard Column, PL1170-1830).

-   -   Volume of injection: 20 μl    -   Solvent composition: [H₂SO₄]: 5.5 mM    -   Temperature of the columns: 65° C.    -   RID (G1362A): temperature set: 35° C.

Acetone was extracted from the filtered aliquots by mixing with methylacetate (1 volume of methyl acetate for 2 volumes of filtered aliquot).Acetone concentration was determined by gas chromatography using Gaschromatograph 450-GC (Bruker) and the following program:

-   -   Column: DB-WAX (123-7033, Agilent Technologies)    -   Injector:        -   Split ratio: 10        -   T°=250° C.    -   Oven:        -   50° C. for 9 minutes        -   20° C. per minute until 180° C.        -   180° C. for 5 minutes        -   Column flow: 1.5 ml/minute (Nitrogen)    -   Detector FID: T°=300° C.

Results

The ratio [acetone] produced (mM)/[glucose] consumed (mM) was higher forthe strain GBE2265 than for the strain GBE2264.

Example 11: Construction of the Strain GBE1283

Strain GBE0929 was plated on MS plates containing glucose as the sourceof carbon (2 g/L). An isolated colony was transferred to MS liquidmedium containing glucose (2 g/L) and incubated 3 days at 30° C. MSliquid medium (100 ml) containing glucose (2 g/L) was inoculated withpre culture of strain GBE0929. The initial OD₆₀₀ was 0.1. The 100 ml ofculture was incubated in a 1 L erlenmeyer, at 30° C., 170 rpm of speed.When the OD₆₀₀ was superior to 1, an aliquot of the culture was takenand used as inoculum for a fresh culture (100 ml of culture incubated in1 L erlenmeyer, at 30° C., 170 rpm of speed). Strain GBE0929 wassub-cultured 10 times to obtain strain GBE1283.

Example 12: Construction of the Strain GBE2256

Strain GBE1283 was cultivated in LB medium and GBE1283 cells were madeelectrocompetent. Electrocompetent GBE1283 cells were transformed withthe pKD46 plasmid and then plated on LB plates containing ampicilline(100 ug/ml). Plates were incubated overnight at 30° C. Transformation ofGBE1283 cells with plasmid pKD46 generated strain GBE1284.

The plasmid pGBE0688 was used as a template along with primers 0629 and0630 (given as SEQ RC0007 and RC0008, respectively) to generate a 1.2Kbp PCR product. The resulting 1.2 Kbp PCR product was transformed intoelectrocompetent GBE1284 bacteria and the transformation mixture wasplated on LB plates containing spectinomycin (50 ug/ml). Plates werethen incubated overnight at 37° C. to generate a new strain namedGBE2252_pKD46. In Strain GB2252_pKD46 the phosphofructokinase gene pfkA,was deleted and was replaced by the spectinomycin resistance cassette.To check that the deletion of the pfkA gene occurred, a PCRamplification was performed with primers 0619 and 0620 (given as SEQRC0009 and RC0010, respectively). This 1.7 Kbp PCR product was sequencedwith the same primers 0619 and 0620. In order to check the loss of theplasmid pKD46, the strain GBE2252_pKD46 was plated on LB plates andincubated overnight at 42° C. The loss of the plasmid pKD46 was verifiedby plating isolated colonies on LB plates containing ampicilline (100ug/ml), incubated overnight at 30° C., and on LB plates incubatedovernight at 37° C. The resulting strain grew on LB plates incubated at37° C. and was named GBE2252. GBE2252 cells did not grow on LB platessupplied with ampicilline (100 ug/ml).

Strain GBE2252 was made electrocompetent, and GBE2252 electrocompetentcells were transformed with plasmid pKD46. Transformant cells were thenplated on LB plates containing ampicilline (100 ug/ml) and plates wereincubated overnight at 30° C. to obtain a new strain named GBE2253.

A PCR product was generated by using the plasmid pGBE0687 as a templateand the oligonucleotides 0631 and 0632 (given as SEQ RC0011 and RC0012,respectively) as primers. The resulting 1.3 Kbp PCR product wastransformed into electrocompetent GBE2253 bacteria and thetransformation mixture was plated on LB plates containing apramycin (50ug/ml). Plates were incubated overnight at 37° C. to generate strainGBE2256_pKD46. In Strain GBE2256_pKD46 the phosphofructokinase genepfkB, was deleted and the deleted DNA sequence was replaced by acassette containing the apramycin resistance gene. To check that thedeletion of the pfkB gene occurred, a PCR amplification was performedwith primers 0621 and 0622 (given as SEQ RC0013 and RC0014,respectively). This final 2.2 Kbp PCR product was sequenced by using thesame primers 0621 and 0622.

In order to induce the loss of the plasmid pKD46, strain GBE2256_(—)pKD46 was plated on LB plates and plates were incubated overnight at 42°C. The loss of the plasmid pKD46 was checked by plating isolatedcolonies on LB plates supplied with ampicilline (100 ug/ml), incubatedovernight at 30° C., and on LB plates incubated overnight at 37° C. Theresulting strain growing on LB plates incubated at 37° C. was namedGBE2256. GBE2256 cells did not grow on LB plates supplied withampicilline (100 ug/ml).

Example 13: Construction of the Strain GBE1518

Plasmid pGBE0688 was used as a template with primers 0633 and 1109(given as SEQ RC0003 and RC0047, respectively) to generate a 1.3 Kbp PCRproduct. This 1.3 Kbp PCR product was transformed into electrocompetentGBE1284 bacteria and the transformation mixture was then plated on LBplates containing spectinomycin (50 ug/ml) and incubated overnight at37° C. to generate strain GBE1433. In strain GBE1433 the DNA sequencecomposed by the zwf gene were deleted. This deleted DNA sequenceincluding the zwf gene was replaced by a spectinomycin resistancecassette. In order to check the effective deletion of the zwf gene, aPCR amplification was performed with primers 1036 and 1110 (given as SEQRC0005 and RC0048, respectively). A final 1.5 Kbp PCR product wasobtained. This 1.5 Kbp PCR product was sequenced with the same primers1036 and 1110.

Strain GBE1433 was made electrocompetent. GBE1433 electrocompetent cellswere transformed with plasmid pKD46, and then plated on LB platessupplied with ampicilline (100 ug/ml). Plates were incubated overnightat 30° C. Transformation of GBE1433 cells with plasmid pKD46 generatedstrain GBE1436.

The plasmid pGBE0687 was used as a template along with primers 0629 and0630 (given as SEQ RC0007 and RC0008, respectively) to generate a 1.2Kbp PCR product. The resulting 1.2 Kbp PCR product was transformed intoelectrocompetent GBE1436 bacteria and the transformation mixture wasplated on LB plates containing apramycin (50 ug/ml). Plates were thenincubated overnight at 37° C. to generate a new strain namedGBE1441_pKD46. In Strain GB1441_pKD46 the phosphofructokinase gene pfkA,was deleted and was replaced by the apramycin resistance cassette. Tocheck that the deletion of the pfkA gene occurred, a PCR amplificationwas performed with primers 0619 and 0620 (given as SEQ RC0009 andRC0010, respectively). This 1.7 Kbp PCR product was sequenced with thesame primers 0619 and 0620. In order to check the loss of the plasmidpKD46, the strain GBE1441_pKD46 was plated on LB plates and incubatedovernight at 42° C. The loss of the plasmid pKD46 was verified byplating isolated colonies on LB plates containing ampicilline (100ug/ml), incubated overnight at 30° C., and on LB plates incubatedovernight at 37° C. The resulting strain grew on LB plates incubated at37° C. and was named GBE1441. GBE1441 cells did not grow on LB platessupplied with ampicilline (100 ug/ml).

The spectinomycin cassette was located at the corresponding loci of thezwf gene and the apramycin cassette was located at the correspondingloci of the pfkA gene. In order to excise the resistant cassettescontaining the spectinomycin and apramycin resistance genes, the strainGBE1441 was transformed with the plasmid pCP20 to obtain the strainGBE1441_p. After overnight incubation on LB plates containingampicilline (100 ug/ml) at 30° C., isolated colonies were restreaked onLB plates supplied with ampicilline (100 ug/ml) and incubated overnightat 30° C. Isolated colonies were then plated on LB plates and incubatedovernight at 42° C. which caused the loss of the pCP20 plasmid. Then, inorder to check the effective excision of the two resistant cassettes andthe loss of the pCP20 plasmid, isolated colonies were streaked out on LBplates containing spectinomycin (50 ug/ml), incubated overnight at 37°C., on LB plates containing apramycin (50 ug/ml), incubated overnight at37° C., on LB plates containing ampicilline (100 ug/ml), incubatedovernight at 30° C. and on LB plates, incubated overnight at 37° C. Theresulting strain grew on LB plates incubated at 37° C. and was namedGBE1448. GBE1448 cells did not grow on LB plates containingspectinomycin (50 ug/ml), on LB plates containing apramycin (50 ug/ml),and on LB plates supplied with ampicilline (100 ug/ml).

Strain GBE1448 was made electrocompetent, and GBE1448 electrocompetentcells were transformed with plasmid pKD46. Transformant cells were thenplated on LB plates containing ampicilline (100 ug/ml) and plates wereincubated overnight at 30° C. to obtain a new strain named GBE1449. APCR product was generated by using the plasmid pGBE0688 as a templateand the oligonucleotides 0631 and 0632 (given as SEQ RC0011 and RC0012,respectively) as primers. The resulting 1.3 Kbp PCR product wastransformed into electrocompetent GBE1449 bacteria and thetransformation mixture was plated on LB plates containing spectinomycin(50 ug/ml). Plates were incubated overnight at 37° C. to generate strainGBE1518_(—) pKD46. In Strain GBE1518_(—) pKD46 the phosphofructokinasegene pfkB, was deleted and the deleted DNA sequence was replaced by acassette containing the spectinomycin resistance gene. To check that thedeletion of the pfkB gene occurred, a PCR amplification was performedwith primers 0621 and 0622 (given as SEQ RC0013 and RC0014,respectively). This final 2.2 Kbp PCR product was sequenced by using thesame primers 0621 and 0622.

In order to induce the loss of the plasmid pKD46, strain GBE1518_(—)pKD46 was plated on LB plates and plates were incubated overnight at 42°C. The loss of the plasmid pKD46 was checked by plating isolatedcolonies on LB plates supplied with ampicilline (100 ug/ml), incubatedovernight at 30° C., and on LB plates incubated overnight at 37° C. Theresulting strain growing on LB plates incubated at 37° C. was namedGBE1518. GBE1518 cells did not grow on LB plates supplied withampicilline (100 ug/ml).

Example 14: Construction of the Strain GBE1420

Plasmid pGBE0688 was used as a template with primers 0633 and 0634(given as SEQ RC0003 and RC0004, respectively) to generate a 1.3 Kbp PCRproduct. This 1.3 Kbp PCR product was transformed into electrocompetentGBE1284 bacteria and the transformation mixture was then plated on LBplates containing spectinomycin (50 ug/ml) and incubated overnight at37° C. to generate strain GBE1287. In strain GBE1287 the DNA sequencecomposed by the zwf, edd, and eda genes were deleted. This deleted DNAsequence including the zwf, edd, and eda genes was replaced by aspectinomycin resistance cassette. In order to check the effectivedeletion of the zwf, edd, and eda genes, a PCR amplification wasperformed with primers 1036 and 1037 (given as SEQ RC0005 and RC0006,respectively). A final 1.9 Kbp PCR product was obtained. This 1.9 KbpPCR product was sequenced with the same primers 1036 and 1037.

Strain GBE1287 was made electrocompetent. GBE1287 electrocompetent cellswere transformed with plasmid pKD46, and then plated on LB platessupplied with ampicilline (100 ug/ml). Plates were incubated overnightat 30° C. Transformation of GBE1287 cells with plasmid pKD46 generatedstrain GBE1337.

The plasmid pGBE0687 was used as a template along with primers 0629 and0630 (given as SEQ RC0007 and RC0008, respectively) to generate a 1.2Kbp PCR product. The resulting 1.2 Kbp PCR product was transformed intoelectrocompetent GBE1337 bacteria and the transformation mixture wasplated on LB plates containing apramycin (50 ug/ml). Plates were thenincubated overnight at 37° C. to generate a new strain namedGBE1353_pKD46. In Strain GB1353_pKD46 the phosphofructokinase gene pfkA,was deleted and was replaced by the apramycin resistance cassette. Tocheck that the deletion of the pfkA gene occurred, a PCR amplificationwas performed with primers 0619 and 0620 (given as SEQ RC0009 andRC0010, respectively). This 1.7 Kbp PCR product was sequenced with thesame primers 0619 and 0620. In order to check the loss of the plasmidpKD46, the strain GBE1353_pKD46 was plated on LB plates and incubatedovernight at 42° C. The loss of the plasmid pKD46 was verified byplating isolated colonies on LB plates containing ampicilline (100ug/ml), incubated overnight at 30° C., and on LB plates incubatedovernight at 37° C. The resulting strain grew on LB plates incubated at37° C. and was named GBE1353. GBE1353 cells did not grow on LB platessupplied with ampicilline (100 ug/ml).

The spectinomycin cassette was located at the corresponding loci of thezwf_edd_eda genes and the apramycin cassette was located at thecorresponding loci of the pfkA genes. In order to excise the resistantcassettes containing the spectinomycin and apramycin resistance genes,the strain GBE1353 was transformed with the plasmid pCP20 to obtain thestrain GBE1353_p. After overnight incubation on LB plates containingampicilline (100 ug/ml) at 30° C., isolated colonies were restreaked onLB plates supplied with ampicilline (100 ug/ml) and incubated overnightat 30° C. Isolated colonies were then plated on LB plates and incubatedovernight at 42° C. which caused the loss of the pCP20 plasmid. Then, inorder to check the effective excision of the two resistant cassettes andthe loss of the pCP20 plasmid, isolated colonies were streaked out on LBplates containing spectinomycin (50 ug/ml), incubated overnight at 37°C., on LB plates containing apramycin (50 ug/ml), incubated overnight at37° C., on LB plates containing ampicilline (100 ug/ml), incubatedovernight at 30° C. and on LB plates, incubated overnight at 37° C. Theresulting strain grew on LB plates incubated at 37° C. and was namedGBE1368. GBE1368 cells did not grow on LB plates containingspectinomycin (50 ug/ml), on LB plates containing apramycin (50 ug/ml),and on LB plates supplied with ampicilline (100 ug/ml).

Strain GBE1368 was made electrocompetent, and GBE1368 electrocompetentcells were transformed with plasmid pKD46. Transformant cells were thenplated on LB plates containing ampicilline (100 ug/ml) and plates wereincubated overnight at 30° C. to obtain a new strain named GBE1371. APCR product was generated by using the plasmid pGBE0688 as a templateand the oligonucleotides 0631 and 0632 (given as SEQ RC0011 and RC0012,respectively) as primers. The resulting 1.3 Kbp PCR product wastransformed into electrocompetent GBE1371 bacteria and thetransformation mixture was plated on LB plates containing spectinomycin(50 ug/ml). Plates were incubated overnight at 37° C. to generate strainGBE1420_(—) pKD46. In Strain GBE1420_(—) pKD46 the phosphofructokinasegene pfkB, was deleted and the deleted DNA sequence was replaced by acassette containing the spectinomycin resistance gene. To check that thedeletion of the pfkB gene occurred, a PCR amplification was performedwith primers 0621 and 0622 (given as SEQ RC0013 and RC0014,respectively). This final 2.2 Kbp PCR product was sequenced by using thesame primers 0621 and 0622.

In order to induce the loss of the plasmid pKD46, strain GBE1420_(—)pKD46 was plated on LB plates and plates were incubated overnight at 42°C. The loss of the plasmid pKD46 was checked by plating isolatedcolonies on LB plates supplied with ampicilline (100 ug/ml), incubatedovernight at 30° C., and on LB plates incubated overnight at 37° C. Theresulting strain growing on LB plates incubated at 37° C. was namedGBE1420. GBE1420 cells did not grow on LB plates supplied withampicilline (100 ug/ml).

Example 15: Acetone Production by the Strains GBE2268 and GBE2269

Description of Plasmid Transformation into Relevant Strains

The strain GBE1283 was made electrocompetent, and GBE1283electrocompetent cells were transformed with plasmid pGB1021.Transformants were then plated on LB plates containing ampicilline (100ug/ml) and plates were incubated overnight at 30° C. to generate strainGBE2266.

Strain GBE1420 was made electrocompetent, and GBE1420 electrocompetentcells were transformed with the plasmid pGBE1020. Transformants werethen plated on LB plates supplied with ampicilline (100 ug/ml). Plateswere incubated overnight at 30° C. to obtain strain GBE2267.

Isolated colonies from strains GBE2266 and GBE2267 were screened on MSplates containing glucose as the source of carbon (2 g/L) andampicilline (100 ug/ml). These plates were incubated at 30° C. to obtainstrains GBE2268 and GBE2269 respectively. After 4 days of incubation at30° C., colonies were transferred to MS liquid medium containing glucose(2 g/L), yeast extract (0.1 g/L) and ampicilline (100 ug/ml) andincubated 3 days at 30° C.

Description of Flasks Conditions

MSP liquid medium (200 ml) containing glucose (10 g/L), yeast extract(0.1 g/L) and ampicilline (100 ug/ml), were inoculated either with preculture of strain GBE2268 or with pre culture of strain GBE2269. Theinitial OD₆₀₀ was 0.1. The 200 ml of culture was incubated in 250 mlbottles, sealed with a screw cap, at 30° C., 170 rpm of speed. Aliquots(2 ml) were taken after 1 day, 2 days, 4 days, 5 days, 6 days, 7 daysand 8 days. For each aliquot sample, the bottle was open for 10 seconds.

Description of Analytical Methods

Aliquots were filtered and the glucose concentration was determined byHPLC analysis using the Agilent HPLC (1260 Infinity) and a Hi-PlexColomn (Agilent, PL1170-6830) with a guard column (Agilent, PL Hi-Plex HGuard Column, PL1170-1830).

-   -   Volume of injection: 20 μl    -   Solvent composition: [H₂SO₄]: 5.5 mM    -   Temperature of the columns: 65° C.    -   RID (G1362A): temperature set: 35° C.        Acetone was extracted from the filtered aliquots by mixing with        methyl acetate (1 volume of methyl acetate for 2 volumes of        filtered aliquot). Acetone concentration was determined by gas        chromatography using Gas chromatograph 450-GC (Bruker) and the        following program:    -   Column: DB-WAX (123-7033, Agilent Technologies)    -   Injector:        -   Split ratio: 10        -   T°=250° C.    -   Oven:        -   50° C. for 9 minutes        -   20° C. per minute until 180° C.        -   180° C. for 5 minutes        -   Column flow: 1.5 ml/minute (Nitrogen)    -   Detector FID: T°=300° C.

Results

The ratio [acetone] produced (mM)/[glucose] consumed (mM) was higher forthe strain GBE2269 than for the strain GBE2268.

Example 16: Acetone Production by the Strains GBE2272 and GBE2273

Description of Plasmid Transformation into Relevant Strains

The strain GBE2256 was made electrocompetent, and GBE2256electrocompetent cells were transformed with plasmid pGB1020.Transformants were then plated on LB plates containing ampicilline (100ug/ml) and plates were incubated overnight at 30° C. to generate strainGBE2270.

Strain GBE1518 was made electrocompetent, and GBE1518 electrocompetentcells were transformed with the plasmid pGBE1020. Transformants werethen plated on LB plates supplied with ampicilline (100 ug/ml). Plateswere incubated overnight at 30° C. to obtain strain GBE2271.

Isolated colonies from strains GBE2270 and GBE2271 were screened on MSplates containing glucose as the source of carbon (2 g/L) andampicilline (100 ug/ml). These plates were incubated at 30° C. to obtainstrains GBE2272 and GBE2273, respectively. After 4 days of incubation at30° C., colonies were transferred to MS liquid medium containing glucose(2 g/L), yeast extract (0.1 g/L) and ampicilline (100 ug/ml) andincubated 3 days at 30° C.

Description of Flasks Conditions

MSP liquid medium (200 ml) containing glucose (10 g/L), yeast extract(0.1 g/L) and ampicilline (100 ug/ml), were inoculated either with preculture of strain GBE2272 or with pre culture of strain GBE2273. Theinitial OD₆₀₀ was 0.1. The 200 ml of culture was incubated in 250 mlbottles, sealed with a screw cap, at 30° C., 170 rpm of speed. Aliquots(2 ml) were taken after 1 day, 2 days, 4 days, 5 days and 6 days. Foreach aliquot sample, the bottle was open for 10 seconds.

Description of Analytical Methods

Aliquots were filtered and the glucose concentration was determined byHPLC analysis using the Agilent HPLC (1260 Infinity) and a Hi-PlexColomn (Agilent, PL1170-6830) with a guard column (Agilent, PL Hi-Plex HGuard Column, PL1170-1830).

-   -   Volume of injection: 20 μl    -   Solvent composition: [H₂SO₄]: 5.5 mM    -   Temperature of the columns: 65° C.    -   RID (G1362A): temperature set: 35° C.        Acetone was extracted from the filtered aliquots by mixing with        methyl acetate (1 volume of methyl acetate for 2 volumes of        filtered aliquot). Acetone concentration was determined by gas        chromatography using Gas chromatograph 450-GC (Bruker) and the        following program:    -   Column: DB-WAX (123-7033, Agilent Technologies)    -   Injector:        -   Split ratio: 10        -   T°=250° C.    -   Oven:        -   50° C. for 9 minutes        -   20° C. per minute until 180° C.        -   180° C. for 5 minutes        -   Column flow: 1.5 ml/minute (Nitrogen)    -   Detector FID: T°=300° C.

Results

The ratio [acetone] produced (mM)/[glucose] consumed (mM) was higher forthe strain GBE2273 than for the strain GBE2272.

TABLE OF RESULTS II GBE2264 GBE2265 GBE2268 GBE2269 GBE2268 GBE2273GBE2272 GBE2273 PEP dependent glucose + + − − − − − − uptake (ptsHI)Heterologous − + − + − + + + phosphoketolase (pkt) EMPP (pfkAB) + − +− + − − − PPP (zwf) + − + − + − + − EDP (edd eda) + − + − + + + +Heterologous acetone + + + + + + + + pathway (thl ctfAB adc)[acetone]_(produced (mM))/ 0.03 0.21 0.01 0.22 0.01 0.51 0.05 0.51[glucose]_(consumed (mM)) the reported ratio was the maximum observed

The invention claimed is:
 1. A genetically modified prokaryoticmicroorganism comprising the following characteristics: (A) increasedphosphoketolase activity as compared to a non-genetically modifiedmicroorganism; and (B) (1) diminished or inactivated phosphofructokinaseactivity as compared to a non-genetically modified microorganism; or (2)not possessing phosphofructokinase activity; and (C)(1) diminished orinactivated glucose-6-phosphate dehydrogenase activity as compared to anon-genetically modified microorganism; or (2) not possessingglucose-6-phosphate dehydrogenase activity.
 2. The genetically modifiedprokaryotic microorganism of claim 1, wherein the genetically modifiedmicroorganism is E. coli comprising the genotype Δzwf_edd_eda ΔpfkAΔpfkB.
 3. The genetically modified prokaryotic microorganism of claim 1,wherein the genetically modified microorganism is E. coli.
 4. Thegenetically modified prokaryotic microorganism of claim 1, wherein thenon-genetically modified microorganism does not have phosphoketolaseactivity.
 5. The genetically modified prokaryotic microorganism of claim4, wherein the non-genetically modified microorganism is geneticallymodified so as to comprise a nucleotide sequence encoding aphosphoketolase.
 6. The genetically modified prokaryotic microorganismof claim 5, wherein the genetically modified prokaryotic microorganismis further genetically modified by mutation and selection for increasedphosphoketolase activity as compared to the non-genetically modifiedmicroorganism.
 7. The genetically modified prokaryotic microorganism ofclaim 1, wherein the non-genetically modified microorganism hasphosphoketolase activity.
 8. The genetically modified prokaryoticmicroorganism of claim 7, wherein the non-genetically modifiedmicroorganism is genetically modified by mutation and selection forincreased phosphoketolase activity as compared to the non-geneticallymodified microorganism.
 9. The genetically modified prokaryoticmicroorganism of claim 7, wherein the non-genetically modifiedmicroorganism is genetically modified so as to comprise a nucleotidesequence allowing for the increased expression of a phosphoketolase ascompared to the non-genetically modified microorganism.
 10. Thegenetically modified prokaryotic microorganism of claim 9, wherein thenucleotide sequence encodes a phosphoketolase.
 11. The geneticallymodified prokaryotic microorganism of claim 9, wherein the nucleotidesequence comprises a heterologous expression control sequence.
 12. Thegenetically modified prokaryotic microorganism of claim 1, wherein thegenetically modified prokaryotic microorganism is genetically modifiedso as to reduce phosphofructokinase activity as compared to anon-genetically modified microorganism.
 13. The genetically modifiedprokaryotic microorganism of claim 12, wherein a gene encoding aphosphofructokinase is genetically modified so as to reducephosphofructokinase activity as compared to the non-genetically modifiedmicroorganism.
 14. The genetically modified prokaryotic microorganism ofclaim 1, wherein the genetically modified prokaryotic microorganism isgenetically modified so as to inactivate phosphofructokinase activity ascompared to the non-genetically modified microorganism.
 15. Thegenetically modified prokaryotic microorganism of claim 14, wherein agene encoding a phosphofructokinase is inactivated.
 16. The geneticallymodified prokaryotic microorganism of claim 1, wherein the geneticallymodified prokaryotic microorganism is genetically modified so as toreduce glucose-6-phosphate dehydrogenase activity as compared to thenon-genetically modified microorganism.
 17. The genetically modifiedprokaryotic microorganism of claim 16, wherein a gene encoding aglucose-6-phosphate dehydrogenase is genetically modified so as toreduce glucose-6-phosphate dehydrogenase activity as compared to thenon-genetically modified microorganism.
 18. The genetically modifiedprokaryotic microorganism of claim 1, wherein the genetically modifiedprokaryotic microorganism is genetically modified so as to inactivateglucose-6-phosphate dehydrogenase activity as compared to thenon-genetically modified microorganism.
 19. The genetically modifiedprokaryotic microorganism of claim 18, wherein a gene encoding aglucose-6-phosphate dehydrogenase is inactivated.
 20. The geneticallymodified prokaryotic microorganism of claim 1, wherein the geneticallymodified prokaryotic microorganism is genetically modified so as toreduce glyceraldehyde 3-phosphate dehydrogenase activity as compared toa non-genetically modified microorganism.
 21. The genetically modifiedprokaryotic microorganism of claim 20, wherein a gene encoding aglyceraldehyde 3-phosphate dehydrogenase is genetically modified so asto reduce glyceraldehyde 3-phosphate dehydrogenase activity as comparedto the non-genetically modified microorganism.
 22. The geneticallymodified prokaryotic microorganism of claim 1, wherein the geneticallymodified prokaryotic microorganism is genetically modified so as toinactivate glyceraldehyde 3-phosphate dehydrogenase activity as comparedto the non-genetically modified microorganism.
 23. The geneticallymodified prokaryotic microorganism of claim 22, wherein a gene encodinga glyceraldehyde 3-phosphate dehydrogenase is inactivated.
 24. Thegenetically modified prokaryotic microorganism of claim 1, furthercomprising fructose-1,6-bisphosphate phosphatase activity.
 25. Thegenetically modified prokaryotic microorganism of claim 24, wherein saidgenetically modified prokaryotic microorganism has increasedfructose-1,6-bisphosphate phosphatase activity when grown on glucose ascompared to a non-genetically modified microorganism.
 26. Thegenetically modified prokaryotic microorganism of claim 25, wherein saidgenetically modified prokaryotic microorganism has been geneticallymodified to have increased fructose-1,6-bisphosphate phosphataseactivity as compared to a non-genetically modified microorganism whengrown on glucose.
 27. The genetically modified prokaryotic microorganismof claim 26, wherein the genetically modified prokaryotic microorganismis further genetically modified so as to reduce glyceraldehyde3-phosphate dehydrogenase activity as compared to a non-geneticallymodified microorganism.
 28. The genetically modified prokaryoticmicroorganism of claim 27, wherein a gene encoding a glyceraldehyde3-phosphate dehydrogenase is genetically modified so as to reduceglyceraldehyde 3-phosphate dehydrogenase activity as compared to thenon-genetically modified microorganism.
 29. The genetically modifiedprokaryotic microorganism of claim 26, wherein the genetically modifiedprokaryotic microorganism is genetically modified so as to inactivateglyceraldehyde 3-phosphate dehydrogenase activity as compared to thenon-genetically modified microorganism.
 30. The genetically modifiedprokaryotic microorganism of claim 29, wherein a gene encoding aglyceraldehyde 3-phosphate dehydrogenase is inactivated.
 31. Thegenetically modified prokaryotic microorganism of claim 26, wherein saidgenetically modified prokaryotic microorganism has been transformed witha nucleic acid encoding a fructose-1,6-bisphosphate phosphatase so as tohave fructose-1,6-bisphosphate phosphatase activity when grown onglucose as compared to the non-genetically modified microorganism. 32.The genetically modified prokaryotic microorganism of claim 1, whereinsaid genetically modified prokaryotic microorganism is a bacterium. 33.The genetically modified prokaryotic microorganism of claim 1, wherein agene encoding a PEP-dependent PTS transporter has been inactivated. 34.The genetically modified prokaryotic microorganism of claim 1, whereinthe genetically modified prokaryotic microorganism is capable ofconverting acetyl-CoA into acetone.
 35. The genetically modifiedprokaryotic microorganism of claim 1, wherein the genetically modifiedprokaryotic microorganism is capable of converting acetyl-CoA intoisobutene.
 36. The genetically modified prokaryotic microorganism ofclaim 1, wherein the genetically modified prokaryotic microorganism iscapable of converting acetyl-CoA into propene.
 37. The geneticallymodified prokaryotic microorganism of claim 1 wherein the geneticallymodified prokaryotic microorganism is capable of converting glucose intoacetyl-CoA.
 38. A method for producing acetyl-CoA from glucosecomprising: (a) culturing a genetically modified prokaryoticmicroorganism in a suitable media, wherein the prokaryotic microorganismcomprises the following characteristics: (1) increased phosphoketolaseactivity as compared to a non-genetically modified microorganism; and(2) (a) diminished or inactivated phosphofructokinase activity ascompared to a non-genetically modified microorganism; or (b) notpossessing phosphofructokinase activity; and (3) (a) diminished orinactivated glucose-6-phosphate dehydrogenase activity as compared to anon-genetically modified microorganism; or (b) not possessingglucose-6-phosphate dehydrogenase activity; and (b) recovering saidacetyl-CoA.
 39. A method for producing acetone, isobutene, or propenecomprising: (a) culturing a genetically modified prokaryoticmicroorganism in a suitable media, wherein the prokaryotic microorganismcomprises: (1) increased phosphoketolase activity as compared to anon-genetically modified microorganism; and (2) (a) diminished orinactivated phosphofructokinase activity as compared to anon-genetically modified microorganism; or (b) not possessingphosphofructokinase activity; and (3) (a) diminished or inactivatedglucose-6-phosphate dehydrogenase activity as compared to anon-genetically modified microorganism; or (b) not possessingglucose-6-phosphate dehydrogenase activity; and (b) recovering saidacetone, isobutene, or propene from the culture medium.
 40. The methodof claim 39, wherein acetone is recovered.
 41. The method of claim 39,wherein isobutene is recovered.
 42. The method of claim 39, whereinpropene is recovered.